Antifungal Paenibacillus strains, fusaricidin-type compounds, and their use

ABSTRACT

The present invention relates to novel isolated bacterial strains, which are members of the genus Paenibacillus, originally isolated from soil and showing antagonistic activity against a broad range of pathogens and being capable of producing antimicrobial metabolites. It was found that the strains Lu16774 and Lu17007 belong to a novel subspecies named Paenibacillus polymyxa ssp. plantarum while the strain Lu17015 belongs to a novel species which is proposed to be Paenibacillus epiphyticus. The present invention also relates to microbial pesticide compositions comprising at least one of such novel bacterial strains, whole culture broth or a cell-free extract or a fraction thereof or at least one metabolite thereof, and/or a mutant of at least one of said novel bacterial strains having all the identifying characteristics of the respective bacterial strain or whole culture broth, cell-free extract, fraction and/or metabolite of the mutant thereof showing antagonistic activity against plant pathogens. The present invention also relates to a method of controlling or suppressing plant pathogens or preventing plant pathogen infections by applying such composition. The present invention also relates to novel fusaricidin-type compounds which are metabolites produced by the strains of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.15/501,784, filed Feb. 3, 2017, which is a National Stage application ofInternational Application No. PCT/EP2015/067925, filed Aug. 4, 2015.This application also claims priority under 35 U.S.C. § 119 to EuropeanPatent Application No. 14179620.1, filed Aug. 4, 2014.

FIELD OF THE INVENTION

The present invention relates to novel isolated bacterial strains, whichare members of the genus Paenibacillus, originally isolated from soiland showing antagonistic activity against a broad range of pathogens andbeing capable of producing antimicrobial metabolites. The presentinvention also relates to microbial pesticide compositions comprising atleast one of such novel bacterial strains, whole culture broth or acell-free extract or a fraction thereof or at least one metabolitethereof, and/or a mutant of at least one of said novel bacterial strainshaving all the identifying characteristics of the respective bacterialstrain or whole culture broth, cell-free extract, fraction and/ormetabolite of the mutant thereof showing antagonistic activity againstplant pathogens. The present invention also relates to a method ofcontrolling or suppressing plant pathogens or preventing plant pathogeninfections by applying such composition. The present invention alsorelates to novel fusaricidin-type compounds which are metabolitesproduced by the strains of the present invention.

REFERENCE TO SEQUENCE LISTING

This application is filed with a Computer Readable Form of a SequenceListing in accord with 37 C.F.R. § 1.821(c). The text file submitted byEFS, “13779-1333_2017-02-03_Sequence_Listing_ST25,” was created on Feb.1, 2017, has a file size of 37.6 Kbytes, and is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

In the technical field of controlling phytopathogenic fungi affectingplants or crops it is well known to apply active compound compositionscomprising biopesticides, for example selected from bacteria, likespore-forming bacteria, or fungi which are not detrimental to the plantor crop to be treated and which biological control agents may be furthercombined with classical organic chemical antagonists of plant pathogens.

Biopesticides have been defined as a form of pesticides based onmicro-organisms (bacteria, fungi, viruses, nematodes, etc.) or naturalproducts (compounds or extracts from biological sources) (U.S.Environmental Protection Agency:http://www.epa.gov/pesticides/biopesticides/).

Biopesticides are typically created by growing and concentratingnaturally occurring organisms and/or their metabolites includingbacteria and other microbes, fungi, viruses, nematodes, proteins, etc.They are often considered to be important components of integrated pestmanagement (IPM) programmes, and have received much practical attentionas substitutes to synthetic chemical plant protection products (PPPs).

Biopesticides fall into two major classes, microbial and biochemicalpesticides:

-   -   (1) Microbial pesticides consist of bacteria, fungi or viruses        (and often include the metabolites that bacteria and fungi        produce). Entomopathogenic nematodes are also classed as        microbial pesticides, even though they are multi-cellular.    -   (2) Biochemical pesticides are naturally occurring substances        that control pests or provide other crop protection uses as        defined below, but are relatively non-toxic to mammals.

For controlling phytopathogenic fungi several microbial pesticidescomprising spore-forming bacteria such as Bacillus subtilis have beendescribed earlier, see e. g. WO 1998/050422; WO 2000/029426; WO1998/50422 and WO 2000/58442.

WO 2009/0126473 discloses agriculturally acceptable aqueous compositionscomprising bacterial or fungal spores contained in an aqueous/organicsolvent and which may further comprise insect control agents,pesticides, fungicides or combinations thereof. Spores of bacteria ofthe genus Bacillus are a preferred species.

WO 2006/017361 discloses compositions for controlling plant pathogensand comprising at least one beneficial bacterium, at least onebeneficial fungus, at least on nutrient and at least one compound whichextends the effective lifetime of such a composition. The group ofbeneficial bacteria e.a. comprises bacteria of Paenibacillus polymyxaand Paenibacillus durum.

EP-A-1 168 922 relates to compositions for affecting plant growth and/orimparting disease resistance comprising at least two plant-growthpromoting Rhizobacteria strains and a chitinous compound, wherein saidstrains are selected from the genera Bacillus, Paenibacillus,Brevibacillus, Virgibacillus, Alicyclobacillus, and Aneurinibacillus. Noparticular Paenibacillus strains are, however, exemplified in support ofthe claimed combinations.

WO 1999/059412 discloses a Paenibacillus polymyxa strain PKB1 (bearingATCC accession no. 202127) active against several phytopathogenic fungi.

WO 2006/016558 discloses Paenibacillussp. strains BS-0048, BS-0074,BS-0277 and P. polymyxa strain BS-0105 as well as fusaricidin A andfusaricidin B for protection of plants from infections with fungi. Afurther antifungal Paenibacillus strain BRF-1 has been isolated fromsoybean rhizosphere (African J. Microbiol. Res. 4(24), 2692-2698, 2010).

WO 2011/069227 discloses a P. polymyxa strain JB05-01-1 (bearing ATCCaccession no. PTA-10436) having a highly inhibitory effect againstpathogenic bacteria, pre-dominantly foodborne human pathogenic bacteria.

Budi et al. (Appl Environ Microbiol, 1999, 65, 5148-5150) have isolatedPaenibacillus sp. strain B2 from mycorrhizosphere of Sorghum bicolorhaving antagonistic activity towards soil borne fungal pathogens likePhytophthora parasitica.

A Paenibacillus peoriae strain 11.D.3 isolated by Delaporte, B. (LabCytol Veg, Paris, France) and deposited in the open collection ofAgricultural Research Service, USDA, U.S.A. under the NRRL Accession No.BD-62 (Int. J. Syst Bacteriol. 46(4), 988-1003, 1996, hereinafter alsoreferred to as strain BD-62) from soil in Cote d'Ivoire showedantifungal activity against several phytopathogenic bacteria and fungi(J. Appl. Microbiol. 95, 1143-1151, 2003). NRRL is the abbreviation forthe Agricultural Research Service Culture Collection, an internationaldepositary authority for the purposes of deposing microorganism strainsunder the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THEDEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE, havingthe address National Center for Agricultural Utilization Research,Agricultural Research Service, U.S. Department of Agriculture, 1815North University Street, Peoria, Ill. 61604, USA.

The antimicrobial activity of numerous Paenibacillus strains, i. a. a P.peoriae strain, against numerous bacterial, fungal and yeast pathogenshas been reported elsewhere (Lett. Appl. Microbiol. 43, 541-547, 2006).

Raza et al. (Brazilian Arch. Biol. Techol. 53, 1145-1154, 2010; Eur. J.Plant Pathol. 125: 471-483, 2009) described a fusaricidin-typecompound-producing Paenibacillus polymyxa strain SQR-21 effectiveagainst Fusarium oxysporum.

Fusaricidins are a group of antibiotics isolated from Paenibacillusspp., which belong to the class of cyclic lipodepsipeptides. Theircommon structural features which are conserved throughout the family areas follows: a macrocyclic ring consisting of 6 amino acid residues,three of which are L-Thr, D-allo-Thr and D-Ala, as well as the15-guanidino-3-hydroxypentadecanoic acid tail attached to the N-terminalL-Thr residue by an amide bond (ChemMedChem 7, 871-882, 2012; J.Microbiol. Meth. 85, 175-182, 2011, Table 1 herein). These compounds arecyclized by a lactone bridge between the N-terminal L-Thr hydroxyl groupand the C-terminal D-Ala carbonyl group. The position of the amino acidresidues within the depsipeptide cycle are usually numbered startingwith the abovementioned L-Thr which itself also carries the GHPD chainand ending with the C-terminal D-Ala. Non-limiting examples offusaricidins isolated from Paenibacillus are designated LI-F03, LI-F04,LI-F05, LI-F07 and LI-F08 (J. Antibiotics 40(11), 1506-1514, 1987;Heterocycles 53(7), 1533-1549, 2000; Peptides 32, 1917-1923, 2011) andfusaricidins A (also called LI-F04a), B (also called LI-F04b), C (alsocalled LI-F03a) and D (also called LI-F03b) (J. Antibiotics 49(2),129-135, 1996; J. Antibiotics 50(3), 220-228, 1997). The amino acidchain of a fusaricidin is not ribosomally generated but is generated bya non-ribosomal peptide synthetase. Structural formulae of knownfusaricidins are shown in Table 1 (Biotechnol Lett. 34, 1327-1334, 2012;FIG. 1 therein). The compounds designated as LI-F03a, LI-F03b up toLI-F08a and LI-F08b are herein also referred to as fusaricidins LI-F03a,LI-F03b up to LI-F08a and LI-F08b due to their structure within thefusaricidin family (see Table 1).

TABLE 1 Structures of the fusaricidin family. Fusaricidin X² X³ X⁵ A(LI-F04a) D-Val L-Val D-Asn B (LI-F04b) D-Val L-Val D-Gln C (LI-F03a)D-Val L-Tyr D-Asn D (LI-F03b) D-Val L-Tyr D-Gln LI-F05a D-Val L-IleD-Asn LI-F05b D-Val L-Ile D-Gln LI-F06a D-allo-Ile L-Val D-Asn LI-F06bD-allo-Ile L-Val D-Gln LI-F07a D-Val L-Phe D-Asn LI-F07b D-Val L-PheD-Gln LI-F08a D-Ile L-allo-Ile D-Asn LI-F08b D-Ile L-allo-Ile D-Gln

-   -   wherein an arrow defines a single (amide) bond either between        the carbonyl moiety of GHPD and the amino group of L-Thr        (L-threonine) or between the carbonyl group of one amino acid        and the amino group of a neighboring amino acid, wherein the tip        of the arrow indicates the attachment to the amino group of said        amino acid L-Thr or of said neighboring amino acid; and    -   wherein the single line (without an arrow head) defines a single        (ester) bond between the carbonyl group of D-Ala (D-alanine) and        the hydroxyl group of L-Thr; and wherein GHPD is        15-guanidino-3-hydroxypentadecanoic acid.

Among isolated fusaricidin antibiotics, fusaricidin A has shown the mostpromising antimicrobial activity against a variety of clinicallyrelevant fungi and gram-positive bacteria such a Staphylococcus aureus(MIC value range: 0.78-3.12 μg/ml) (ChemMedChem 7, 871-882, 2012). Thesynthesis of fusaricidin analogues that contain 12-guanidino-dodecanoicacid (12-GDA) or 12-aminododecanoic acid (12-ADA) instead of naturallyoccurring GHPD has been established but the replacement of GHPD by12-ADA resulted in complete loss of the antimicrobial activity while thereplacement of GHPD by 12-GDA retained antimicrobial activity(Tetrahedron Lett. 47, 8587-8590, 2006; ChemMedChem 7, 871-882, 2012).

Fusaricidins A, B, C and D are also reported to inhibit plant pathogenicfungi such as Fusarium oxysporum, Aspergillus niger, Aspergillus oryzae,and Penicillum thomii (J. Antibiotics 49(2), 129-135, 1996; J.Antibiotics 50(3), 220-228, 1997). Fusaricidins such as LI-F05, LI-F07and LI-F08 have been found to have certain antifungal activity againstvarious plant pathogenic fungi such as Fusarium moniliforme, F.oxysporum, F. roseum, Giberella fujkuroi, Helminthosporium sesamum andPenicillium expansum (J. Antibiotics 40(11), 1506-1514, 1987).Fusaricidins also have antibacterial activity to Gram-positive bacteriaincluding Staphylococcus aureus (J. Antibiotics 49, 129-135, 1996; J.Antibiotics 50, 220-228, 1997). In addition, fusaricidins haveantifungal activity against Leptosphaeria maculans which causes blackroot rot of canola (Can. J. Microbiol. 48, 159-169, 2002). Moreover,fusaricidins A and B and two related compounds thereof, whereinD-allo-Thr is bound via its hydroxyl group to an additional alanineusing an ester bridge, produced by certain Paenibacillus strains werefound to induce resistance reactions in cultured parsley cells and toinhibit growth of Fusarium oxysporum (WO 2006/016558; EP 1 788 074 A1).

WO 2007/086645 describes the fusaricidin synthetase enzyme and itsencoding gene as isolated from Paenibacillus polymyxa strain E681 whichenzyme is involved in the synthesis of fusaricidins A, B, C, D, LI-F03,LI-F04, LI-F05, LI-F07 and LI-F08.

The genome of several Paenibacillus polymyxa strains has been publishedso far: inter alia for strain M-1 (NCBI acc. no. NC_017542; J.Bacteriol. 193 (29), 5862-63, 2011; BMC Microbiol. 13, 137, 2013),strain CR1 (GenBank acc. no. CP006941; Genome Announcements 2 (1), 1,2014) and strain SC2 (GenBank acc. nos. CP002213 and CP002214; NCBI acc.no. NC_014622; J. Bacteriol. 193 (1), 311-312, 2011), for furtherstrains see legend of FIG. 12 herein. The P. polymyxa strain M-1 hasbeen deposited in China General Microbiological Culture CollectionCenter (CGMCC) under acc. no. CGMCC 7581.

Montefusco et al. describe in Int. J. Systematic Bacteriol. (43,388-390, 1993) a novel bacterial species of the genus Bacillus andsuggest the name Bacillus peoriae which may be distinguished from otherBacillus strains as for example Bacillus badius, B. coagulans, B.polymyxa and others. Said novel Bacillus strain is reported to producespores, to be gram-positive and to produce catalase, without producingoxidase. Further biochemical characteristics are summarized therein. Thestrain, which may be isolated from soil or rotting vegetable materials,was designated BD-57 and was deposited at the Agricultural ResearchService, USDA, U.S.A. as NRRL B-14750 and also at the DSMZ (see below)as strain DSM 8320. Based on further biochemical and genetic analysissaid strain later has been renamed as Paenibacillus peoriae (see Int. J.Systematic Bacteriol. 46, 988-1003, 1996). A more recent assessment ofthe diversity of Paenibacillus spp. in the maize rhizosphere usingPCR-DGGE method was described in J. Microbiol. Methods 54, 213-231,2003.

Biopesticides for use against crop diseases have already establishedthemselves on a variety of crops. For example, biopesticides alreadyplay an important role in controlling downy mildew diseases. Theirbenefits include: a 0-Day Pre-Harvest Interval and the ability to useunder moderate to severe disease pressure.

A major growth area for biopesticides is in the area of seed treatmentsand soil amendments. Biopesticidal seed treatments are e. g. used tocontrol soil borne fungal pathogens that cause seed rots, damping-off,root rot and seedling blights. They can also be used to control internalseed borne fungal pathogens as well as fungal pathogens that are on thesurface of the seed. Many biopesticidal products also show capacities tostimulate plant host defenses and other physiological processes that canmake treated crops more resistant to a variety of biotic and abioticstresses.

However, biopesticides under certain conditions can also havedisadvantages, such as high specificity (requiring an exactidentification of the pest/pathogen and the use of multiple products),slow speed of action (thus making them unsuitable if a pest outbreak isan immediate threat to a crop), variable efficacy due to the influencesof various biotic and abiotic factors (since biopesticides are usuallyliving organisms, which bring about pest/pathogen control by multiplyingwithin the target insect pest/pathogen), and resistance development.

Therefore there is a need for further bacterial strains and for furtherantimicrobial metabolites which antagonize phytopathogenicmicroorganisms, in particular fungi, which are characterized by a broadspectrum of activity against all classes of phytopathogenic fungi.

DESCRIPTION OF THE INVENTION

Said problem was, surprisingly solved by providing novel strains ofbacteria of the genus Paenibacillus which are characterized by a uniqueprofile of antagonistic activity against phytopathogenic fungi, alsoextending to plant leaf pathogens, as for example selected fromAlternaria spp., Botrytis cinerea, Phytophthora infestans, andSclerotinia sclerotiorum. Said bacterial strains have been depositedwith the International Depositary Authority: Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH, Inhoffenstraße 7 B, 38124Braunschweig, Germany (hereinafter DSMZ).

Furthermore, the whole culture broth, the culture medium and cell-freeextracts of these bacterial strains showed inhibitory activity at leastagainst Alternaria spp., Botrytis cinerea and Phytophthora infestans.Bioactivity guided fractionation of organic extracts led to theisolation of two novel fusaricidin-type compounds (compounds 1A and 1B),the structure of which were elucidated by 1D- and 2D-NMR spectroscopy aswell as mass spectrometry.

Thus, the present invention relates to an isolated microorganism, beinga member of the family Paenibacillus, having at least one of theidentifying characteristics of one of the following strains:

-   -   1) Paenibacillus sp. strain Lu16774 deposited with DSMZ under        Accession No. DSM 26969,    -   2) Paenibacillus sp. strain Lu17007 deposited with DSMZ under        Accession No. DSM 26970, and    -   3) Paenibacillus sp. strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971.

As used herein, the term Paenibacillus sp. strain is identical to theterm Paenibacillus strain.

As used herein, “isolate” refers to a pure microbial culture separatedfrom its natural origin, such an isolate obtained by culturing a singlemicrobial colony. An isolate is a pure culture derived from aheterogeneous, wild population of microorganisms.

As used herein, “strain” refers to isolate or a group of isolatesexhibiting phenotypic, physiological, metabolic and/or genotypic traitsbelonging to the same lineage, distinct from those of other isolates orstrains of the same species.

A further embodiment relates to a whole culture broth, a supernatant ora cell-free extract or a fraction or at least one metabolite of at leastone of the microorganisms as defined above which preferably exhibitantagonistic activity against at least one plant pathogen.

As used herein, “whole culture broth” refers to a liquid culture of amicroorganism containing vegetative cells and/or spores suspended in theculture medium and optionally metabolites produced by the respectivemicroorganism.

As used herein, “culture medium”, refers to a medium obtainable byculturing the microorganism in said medium, preferably a liquid broth,and remaining when cells grown in the medium are removed, e. g., thesupernatant remaining when cells grown in a liquid broth are removed bycentrifugation, filtration, sedimentation, or other means well known inthe art; comprising e. g. metabolites produced by the respectivemicroorganism and secreted into the culture medium. The “culture medium”sometimes also referred to as “supernatant” can be obtained e. g. bycentrifugation at temperatures of about 2 to 30° C. (more preferably attemperatures of 4 to 20° C.) for about 10 to 60 min (more preferablyabout 15 to 30 min) at about 5,000 to 20,000×g (more preferably at about15,000×g).

As used herein, “cell-free extract” refers to an extract of thevegetative cells, spores and/or the whole culture broth of amicroorganism comprising cellular metabolites produced by the respectivemicroorganism obtainable by cell disruption methods known in the artsuch as solvent-based (e. g. organic solvents such as alcohols sometimesin combination with suitable salts), temperature-based, application ofshear forces, cell disruption with an ultrasonicator. The desiredextract may be concentrated by conventional concentration techniquessuch as drying, evaporation, centrifugation or alike. Certain washingsteps using organic solvents and/or water-based media may also beapplied to the crude extract preferably prior to use.

As used herein, the term “metabolite” refers to any component, compound,substance or byproduct (including but not limited to small moleculesecondary metabolites, polyketides, fatty acid synthase products,non-ribosomal peptides, ribosomal peptides, proteins and enzymes)produced by a microorganism (such as fungi and bacteria, in particularthe strains of the invention) that has any beneficial effect asdescribed herein such as pesticidal activity or improvement of plantgrowth, water use efficiency of the plant, plant health, plantappearance, or the population of beneficial microorganisms in the soilaround the plant activity herein.

As used herein, “isolate” refers to a pure microbial culture separatedfrom its natural origin, such an isolate obtained by culturing a singlemicrobial colony. An isolate is a pure culture derived from aheterogeneous, wild population of microorganisms.

As used herein, “strain” refers to isolate or a group of isolatesexhibiting phenotypic and/or genotypic traits belonging to the samelineage, distinct from those of other isolates or strains of the samespecies.

A further embodiment relates to novel compounds of formula I

wherein

-   R is selected from 15-guanidino-3-hydroxypentadecanoic acid (GHPD)    and 12-guanidinododecanoic acid (12-GDA);-   X¹ is threonine;-   X² is isoleucine;-   X³ is tyrosine;-   X⁴ is threonine;-   X⁵ is selected from glutamine and asparagine;-   X⁶ is alanine; and    wherein an arrow defines a single (amide) bond either between the    carbonyl moiety of R and the amino group of the amino acid X¹ or    between the carbonyl group of one amino acid and the amino group of    a neighboring amino acid, wherein the tip of the arrow indicates the    attachment to the amino group of said amino acid X¹ or of said    neighboring amino acid; and    wherein the single line (without an arrow head) defines a single    (ester) bond between the carbonyl group of X⁶ and the hydroxyl group    of X¹;    and the agriculturally acceptable salts thereof, and to methods of    preparing compounds of formula I of the invention which method    comprises culturing the strains of the invention and isolating said    compounds of formula I from the whole culture broth.

According to a further embodiment, the invention further relates tocompounds 1A and 1B, which are of formula I, wherein R is GHPD andwherein X⁵ is asparagine in case of compound 1A and X⁵ is glutamine incase of compound 1B:

The present invention further relates to compositions comprising thestrains, whole culture broth, cell-free extracts, culture media, orcompounds of formula I and their salts of the invention, as well as totheir use for controlling or suppressing plant pathogens or preventingplant pathogen infection or for protection of materials againstinfestation destruction by harmful microorganisms, and to correspondingmethods which comprise treating the pathogens, their habitat or thematerials or plants to be protected against pathogen attack, or the soilor propagation material with an effective amount of the compositions,strains, whole culture broth, cell-free extracts, culture media, orcompounds of formula I and their salts of the invention.

Further embodiments of the invention are disclosed in the followingdetailed description of the invention, the claims and the figures.

The invention relates to the microorganism strains

-   -   1) Paenibacillussp. strain Lu16774 deposited with DSMZ under        Accession No. DSM 26969,    -   2) Paenibacillussp. strain Lu17007 deposited with DSMZ under        Accession No. DSM 26970, and    -   3) Paenibacillus sp. strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971.

The strains Lu16774, Lu 17007 and Lu17015 have been isolated from soilsamples from a variety of European locations including Germany anddeposited under the Budapest Treaty with the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ) under the above-mentionedAccession numbers on Feb. 20, 2013 by BASF SE, Germany.

The genus Paenibacillus (formerly rRNA group 3 bacilli) has beencharacterized phenotypically and physiologically (Antonie vanLeeuwenhoek 64, 253-260 (1993)) by:

-   -   rod-shaped cells of Gram-positive structure,    -   weak reaction with Gram's stain, often even stain negatively,    -   differentiation into ellipsoidal endospores which distinctly        swell the sporangium (mother cell),    -   facultative anaerobic growth with strong growth in absence of        air irrespective of whether nitrate is present or not,    -   fermentation of a variety of sugars,    -   acid and gas formation from various sugars including glucose,    -   no acid production from adonitol and sorbitol,    -   Urease-negative (with exception of P. validus),    -   arginine dihydrolase negative,    -   no utilization of citrate,    -   no growth in presence of 10% sodium chloride,    -   secretion of numerous extracellular hydrolytic enzymes degrading        DNA, protein, starch; and/or    -   G+C content of DNA from 40% to 54%.

The genus Paenibacillus (formerly rRNA group 3 bacilli) has also beencharacterized by 16S rDNA analysis (Antonie van Leeuwenhoek 64, 253-260(1993)):

-   -   having a specific 22-base sequence in a variable region V5 of        the 16S rDNA (5′ to 3′): TCGATACCCTTGGTGCCGAAGT (Antonie van        Leeuwenhoek 64, 253-260 (1993), see Table 3 therein); and/or    -   by hybridization of isolated or PCR-amplified chromosomal DNA        with BG3 probe (5′-TCGATACCCTTGGTGCCGAAGT-3′) (see Antonie van        Leeuwenhoek 64, 253-260 (1993)).

The deposited strains Lu16774, Lu17007 and Lu17015 of the invention weredetermined to belong to the genus Paenibacillus on the followingmorphological and physiological observations (see Example 2.3 herein):

-   -   rod-shaped cells    -   ellipsoidal spores    -   swollen sporangium    -   anaerobic growth    -   fermentation of a variety of sugars including glucose,        arabinose, xylose, mannit, fructose, raffinose, trehalose and        glycerol with acid formation    -   gas production from glucose    -   arginine dihydrolase negative    -   no utilization of citrate    -   no growth in presence of 5% or more sodium chloride    -   production of extracellular hydrolytic enzymes degrading starch,        gelatine, casein and esculin.

Further, the deposited strains Lu16774, Lu17007 and Lu17015 of theinvention were also determined to belong to the genus Paenibacillus by16S rDNA analysis by having the Paenibacillus specific 22-base sequencein 16S rDNA (5′ to 3′):

5′-TCGATACCCTTGGTGCCGAAGT-3′(see SEQ ID NO:1 (nucleotides 840-861), SEQ ID NO:2 (840-861), SEQ IDNO:3 (844-865) and SEQ ID NO:4 (840-861) in sequence listings herein).

Further, sequencing of the complete 16S rDNA in comparison to 24different Paenibacillus strains resulted in clustering of the depositedstrains Lu16774, Lu17007 and Lu17015 with the type strains ofPaenibacillus brasiliensis, P. kribbensis; P. jamilae, P. peoriae, andP. polymyxa, more preferably to P. peoriae, in particular Paenibacilluspeoriae strain BD-62 (see FIGS. 1 and 2 herein). It is known that P.polymyxa and P. peoriae have 16S rDNA sequence identity values of 99.6to 99.7% (J. Gen. Appl. Microbiol. 48, 281-285 (2002)).

“Percent Identity” or “percent similarity” between two nucleotidesequences means percent identity of the residues over the completelength of the aligned sequences and is determined by comparing twooptimally locally aligned sequences over a comparison window defined bythe length of the local alignment between the two sequences, such as,for example, the identity calculated (for rather similar sequences)after manual alignment with the aid of the program AE2 (Alignment Editor2). Local alignment between two sequences only includes segments of eachsequence that are deemed to be sufficiently similar according to thecriterion that depends on the algorithm used to perform the alignment(e. g. AE2, BLAST, secondary structure of the rRNA molecule or alike).The percentage identity is calculated by determining the number ofpositions at which the identical nucleic acid occurs in both sequencesto yield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100.

To determine the percent sequence identity of two nucleic acid sequences(e. g., one of the nucleotide sequences of Table 1 and a homologthereof), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one nucleic acid foroptimal alignment with the other nucleic acid). The bases atcorresponding positions are then compared. When a position in onesequence is occupied by the base as the corresponding position in theother sequence then the molecules are identical at that position. It isto be understood that for the purposes of determining sequence identitywhen comparing a DNA sequence to an RNA sequence, a thymidine nucleotideis equivalent to a uracil nucleotide.

For alignment, the sequence data was put into the program AE2(http://iubio.bio.indiana.edu/soft/molbio/unix/ae2.readme), alignedmanually according to the secondary structure of the resulting rRNAmolecule and compared with representative 16S rRNA gene sequences oforganisms belonging to the Firmicutes (Nucl. Acids Res. 27, 171-173,1999). To obtain % identity values for multiple sequences, all sequencesof were aligned with each other (multiple sequence alignment). Further,to obtain % identity values between two sequences over a longer stretchof aligned sequences in comparison to multiple alignment, a manualpairwise sequence alignment was done as described above using AE2(pairwise sequence alignment).

Further, standardized, automated ribotyping is performed using theQualicon RiboPrintersystem with the Paenibacillus strains Lu16774,Lu17007 and Lu17015 in comparison to the P. peoriae BD-62 using therestriction enzyme EcoRI resulted in similarity of all three novelstrains to P. peoriae BD-62 of between 0.24 and 0.5 (Example 2.2 andFIG. 12).

In sum, the strains have been designated to the following taxonomicgroups.

The Paenibacillus strains Lu16774 and Lu17007 both belong to the speciesPaenibacillus polymyxa.

Thus, the invention relates to the microorganism strains 1)Paenibacillus polymyxa strain Lu16774 deposited with DSMZ underAccession No. DSM 26969,

-   -   2) Paenibacillus polymyxa strain Lu17007 deposited with DSMZ        under Accession No. DSM 26970, and    -   3) Paenibacillus sp. strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971.

According to the results of the phylogenetic analysis presented herein(FIGS. 12 to 22) and unpublished results of Professor Borriss, Germany,it is proposed that the heterogenous species Paenibacillus polymyxarequires a new taxonomic classification into two subspecies:

1) Paenibacillus polymyxa ssp. polymyxa and 2) Paenibacillus polymyxassp. plantarum; and 3) a novel species Paenibacillus nov. spec.epiphyticus.

The type strain P. polymyxa DSM 36 together with the P. polymyxa strainsSQR-21, CFOS, CICC 10580, NRRL B-30509 and A18 form in each of themaximum likielihood dendrograms analysed for five conserved housekeeping genes (dnaN, gyrB, recA, recN and rpoA) a separate cluster(FIGS. 17-21).

Very similar results have been obtained by determination of the AverageAmino acid Identity (AAI) which is frequently used for determination ofphylogenetic relationship amongst bacterial species. This method isbased on the calculation of the average identity of a core genome onamino acid level (Proc. Natl. Acad. USA 102, 2567-2572, 2005). Accordingto the resulting AAI-matrix in FIG. 22, P. polymyxa DSM 36 formstogether with the P. polymyxa SQR-21 strain a sub cluster that isdifferent from the two other sub clusters shown therein.

The strains Lu16674 and Lu17007 together with strain P. polymyxa M-1,1-43, SC2 and Sb3-1 form the second sub cluster in each of the maximumlikielihood dendrograms analysed for five conserved house keeping genes(dnaN, gyrB, recA, recN and rpoA) (FIGS. 17-21). According to AAI-matrixin FIG. 22 based on the analysis of the core genome, this second subcluster is confirmed by its representative strains Lu16674 and Lu17007together with the P. polymyxa M-1 and SC2 strains.

The difference between the two sub clusters is not so significant tojustify a new species, but the AAI identify levels between therepresentatives of both clusters is of about 97.5% justifying theclassification into two separate subspecies

Thus, it is proposed to nominate the first sub cluster according to thetype P. polymyxa strain DSM 36^(T) Paenibacillus polymyxa ssp. polymyxa.Besides strain DSM 36, the P. polymyxa strains SQR-21, CF05, CICC 10580,NRRL B-30509 and A18 shall belong to the subspecies Paenibacilluspolymyxa ssp. polymyxa.

Further, it is proposed to nominate the second sub cluster as novelsubspecies Paenibacillus polymyxa ssp. plantarum. Besides the strainsLu16674 and Lu17007, the P. polymyxa strains M-1, 1-43, SC2 and Sb3-1shall belong to Paenibacillus polymyxa ssp. plantarum.

The strain Lu17015 has only 94.9% identity (AAI) amongst the genes ofthe core genome with the type strain Paenibacillus polymyxa DSM36=ATCC842 (FIG. 22). Thus, the strain Lu17015 could not have been designatedto the species Paenibacillus polymyxa nor to any other knownPaenibacillus species. Similar values are found for the strains E681(94.7%) and CR2 (94.9%). Amongst each other, these three strains have atleast 98.1% identity (AAI). According to the species definition ofKonstantinides and Tiedje (Proc Natl. Acad. Sci. USA. 102, 2567-2572,2005), the strain Lu17015 and also the strains E681 and CR2 can bedesignated to a novel species. Thus, a new species Paenibacillus spec.nov. epiphyticus is proposed herewith. Consequently, the Paenibacillusstrain Lu17015 belongs to Paenibacillus epiphyticus. It is proposed thatsaid strain shall be the type strain. Likewise, the dendrograms based onthe sequence comparisons of the five house keeping genes (FIGS. 17-21)show that this clauster of distant from all other P. polymyxa strains.Besides Lu17015, it is proposed that the P. polymyxa strains E681, CR2TD94, DSM 365 and WLY78 shall belong to Paenibacillus spec. nov.epiphyticus.

Thus, the invention relates to the microorganism strains

-   -   4) Paenibacillus polymyxa ssp. plantarum strain Lu16774        deposited with DSMZ under Accession No. DSM 26969,    -   5) Paenibacillus polymyxa ssp. plantarum strain Lu17007        deposited with DSMZ under Accession No. DSM 26970, and    -   6) Paenibacillus epiphyticus strain Lu17015 deposited with DSMZ        under Accession No. DSM 26971.

In addition to the strains Lu16774, Lu17007 and Lu17015, the inventionrelates to any Paenibacillus strain, whether physically derived from theoriginal deposit of any of the strains Lu16774, Lu17007 and Lu17015 orindependently isolated, so long as they retain at least one of theidentifying characteristics of the deposited Paenibacillus strainsLu16774, Lu17007 and Lu17015. Such Paenibacillus strains of theinvention include any progeny of any of the strains Lu16774, Lu17007 andLu17015, including mutants of said strains.

The term “mutant” refers a microorganism obtained by direct mutantselection but also includes microorganisms that have been furthermutagenized or otherwise manipulated (e. g., via the introduction of aplasmid). Accordingly, embodiments include mutants, variants, and orderivatives of the respective microorganism, both naturally occurringand artificially induced mutants. For example, mutants may be induced bysubjecting the microorganism to known mutagens, such as X-ray, UVradiation or N-methyl-nitrosoguanidine, using conventional methods.Subsequent to said treatments a screening for mutant strains showing thedesired characteristics may be performed.

Mutant strains may be obtained by any methods known in the art such asdirect mutant selection, chemical mutagenesis or genetic manipulation(e. g., via the introduction of a plasmid). For example, such mutantsare obtainable by applying a known mutagen, such as X-ray, UV radiationor N-methyl-nitrosoguanidine. Subsequent to said treatments a screeningfor mutant strains showing the desired characteristics may be performed.

A Paenibacillus strain of the invention is in particular one whichcomprises a DNA sequence exhibiting at least at least 99.6%, preferablyat least 99.8%, even more preferably at least 99.9%, and in particular100.0% nucleotide sequence identity to any one of the 16S rDNA sequencesof the strains Lu16774, Lu17007 and Lu17015, i.e. to any one of thosenucleotide sequences set forth in the Sequence listing being SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NO:3.

According to a further embodiment, a Paenibacillus strain of theinvention is in particular one which comprises a DNA sequence exhibiting100% nucleotide sequence identity to any one of the 16S rDNA sequencesof the strains Lu16774, Lu17007 and Lu17015, i.e. to any one of thosenucleotide sequences set forth in the Sequence listing being SEQ IDNO:1, SEQ ID NO:2 or SEQ ID NO:3.

According to a further embodiment, a Paenibacillus strain of theinvention is one whose complete 16S rDNA sequence has after optimalalignment within the aligned sequence window at least 99.6% identity toat least one of the sequences SEQ ID NO:1 and SEQ ID NO:2 or at least99.8% identity to SEQ ID NO:3; preferably at least 99.8% identity to atleast one of the sequences SEQ ID NO:1, SEQ ID:2 and SEQ ID NO:3; morepreferably at least 99.9% identity to at least one of the sequences SEQID NO:1, SEQ ID NO:2 and SEQ ID NO:3; even more preferably greater than99.9% identity to at least one of the sequences SEQ ID NO:1, SEQ ID:2and SEQ ID NO:3; in particular 100% identity to at least one of thesequences SEQ ID NO:1, SEQ ID:2 and SEQ ID NO:3.

According to a further embodiment, a Paenibacillus strain of theinvention is is selected from the group consisting of:

-   -   a) strain Lu16774 deposited with DSMZ under Accession No. DSM        26969;    -   b) strain Lu17007 deposited with DSMZ under Accession No. DSM        26970;    -   c) strain Lu17015 deposited with DSMZ under Accession No. DSM        26971; and    -   d) a strain which comprises a DNA sequence exhibiting        -   d1) at least 99.6% nucleotide sequence identity to the DNA            sequences SEQ ID NO:4 or SEQ ID NO:9; or        -   d2) at least 99.8% nucleotide sequence identity to the DNA            sequence SEQ ID NO:14; or        -   d3) at least 99.9% nucleotide sequence identity to the DNA            sequences SEQ ID NO:5 or SEQ ID NO:10; or        -   d4) at least 99.2% nucleotide sequence identity to the DNA            sequence SEQ ID NO:15; or        -   d5) at least 99.2% nucleotide sequence identity to the DNA            sequences SEQ ID NO:6 or SEQ ID NO:11; or        -   d6) at least 99.8% nucleotide sequence identity to the DNA            sequence SEQ ID NO:16; or        -   d7) at least 99.8% nucleotide sequence identity to the DNA            sequences SEQ ID NO:7 or SEQ ID NO:12; or        -   d8) at least 99.3% nucleotide sequence identity to the DNA            sequence SEQ ID NO:17; or        -   d9) 100.0% nucleotide sequence identity to the DNA sequences            SEQ ID NO:8 or SEQ ID NO:13; or        -   d10) at least 99.8% nucleotide sequence identity to the DNA            sequence SEQ ID NO:18.

A Paenibacillus strain of the invention is in particular one whichcomprises a dnaN DNA sequence exhibiting at least 99.6% nucleotidesequence identity to the DNA sequences SEQ ID NO:4 or SEQ ID NO:9 orwhich comprises a DNA sequence exhibiting at least 99.8% nucleotidesequence identity to the DNA sequence SEQ ID NO:14.

According to a further embodiment, a Paenibacillus strain of theinvention is one whose complete dnaN DNA sequence has after optimalalignment within the aligned sequence window at least 99.6% identity toat least one of the DNA sequences SEQ ID NO:4 and SEQ ID NO:9 or atleast 99.8% identity to SEQ ID NO:14; preferably at least 99.9% identityto SEQ ID NO:14; in particular 100% identity to SEQ ID NO:14.

A Paenibacillus strain of the invention is in particular one whichcomprises a DNA sequence exhibiting at least 99.8%, in particular 100.0%nucleotide sequence identity to any one of the dnaN DNA sequences of thestrains Lu16774, Lu17007 and Lu17015, i.e. to any one of those DNAsequences SEQ ID NO:4, SEQ ID NO:9 and SEQ ID NO:14.

A Paenibacillus strain of the invention is in particular one whichcomprises a gyrB DNA sequence exhibiting at least 99.9% nucleotidesequence identity to the DNA sequences SEQ ID NO:5 or SEQ ID NO:10 orwhich comprises a DNA sequence exhibiting at least 99.2% nucleotidesequence identity to the DNA sequence SEQ ID NO:15.

According to a further embodiment, a Paenibacillus strain of theinvention is one whose complete gyrB DNA sequence has after optimalalignment within the aligned sequence window at least 99.9% identity toat least one of the DNA sequences SEQ ID NO:5 and SEQ ID NO:10 or atleast 99.9% identity to SEQ ID NO:15; preferably at least 99.9% identityto SEQ ID NO:15; in particular 100% identity to SEQ ID NO:15.

A Paenibacillus strain of the invention is in particular one whichcomprises a DNA sequence exhibiting 100.0% nucleotide sequence identityto any one of the gyrB DNA sequences of the strains Lu16774, Lu17007 andLu17015, i.e. to any one of those DNA sequences SEQ ID NO:5, SEQ IDNO:10 and SEQ ID NO:15.

A Paenibacillus strain of the invention is in particular one whichcomprises a recF DNA sequence exhibiting at least 99.2% nucleotidesequence identity to the DNA sequences SEQ ID NO:6 or SEQ ID NO:11 orwhich comprises a DNA sequence exhibiting at least 99.8% nucleotidesequence identity to the DNA sequence SEQ ID NO:16.

According to a further embodiment, a Paenibacillus strain of theinvention is one whose complete recF DNA sequence has after optimalalignment within the aligned sequence window at least 99.2% identity toat least one of the DNA sequences SEQ ID NO:6 and SEQ ID NO:11 or atleast 99.8% identity to SEQ ID NO:16; preferably at least 99.9% identityto SEQ ID NO:16; in particular 100% identity to SEQ ID NO:16.

A Paenibacillus strain of the invention is in particular one whichcomprises a DNA sequence exhibiting at least 99.8%, in particular 100.0%nucleotide sequence identity to any one of the recF DNA sequences of thestrains Lu16774, Lu17007 and Lu17015, i.e. to any one of those DNAsequences SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:16.

A Paenibacillus strain of the invention is in particular one whichcomprises a recN DNA sequence exhibiting at least 99.8% nucleotidesequence identity to the DNA sequences SEQ ID NO:7 or SEQ ID NO:12 orwhich comprises a DNA sequence exhibiting at least 99.3% nucleotidesequence identity to the DNA sequence SEQ ID NO:17.

According to a further embodiment, a Paenibacillus strain of theinvention is one whose complete recN DNA sequence has after optimalalignment within the aligned sequence window at least 99.8% identity toat least one of the DNA sequences SEQ ID NO:7 and SEQ ID NO:12 or atleast 99.3% identity to SEQ ID NO:17; preferably at least 99.6% identityto SEQ ID NO:17; in particular 100% identity to SEQ ID NO:17.

A Paenibacillus strain of the invention is in particular one whichcomprises a DNA sequence exhibiting at least 99.8%, in particular 100.0%nucleotide sequence identity to any one of the recN DNA sequences of thestrains Lu16774, Lu17007 and Lu17015, i.e. to any one of those DNAsequences SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:17.

A Paenibacillus strain of the invention is in particular one whichcomprises a rpoA DNA sequence exhibiting 100.0% nucleotide sequenceidentity to the DNA sequences SEQ ID NO:8 or SEQ ID NO:13 or whichcomprises a DNA sequence exhibiting at least 99.8% nucleotide sequenceidentity to the DNA sequence SEQ ID NO:18.

According to a further embodiment, a Paenibacillus strain of theinvention is one whose complete rpoA DNA sequence has after optimalalignment within the aligned sequence window 100.0% identity to at leastone of the DNA sequences SEQ ID NO:8 and SEQ ID NO:13 or at least 99.8%identity to SEQ ID NO:18; preferably at least 99.9% identity to SEQ IDNO:17; in particular 100% identity to SEQ ID NO:18.

A Paenibacillus strain of the invention is in particular one whichcomprises a DNA sequence exhibiting 100.0% nucleotide sequence identityto any one of the rpoA DNA sequences of the strains Lu16774, Lu17007 andLu17015, i.e. to any one of those DNA sequences SEQ ID NO:8, SEQ IDNO:13 and SEQ ID NO:18.

A further embodiment relates to an isolated microorganism, being amember of the family Paenibacillus, having at least one of theidentifying characteristics of one of the following strains:

-   -   1) Paenibacillus strain Lu16774 deposited with DSMZ under        Accession No. DSM 26969,    -   2) Paenibacillus strain Lu17007 deposited with DSMZ under        Accession No. DSM 26970, or    -   3) Paenibacillus strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971.

A further embodiment relates to a Paenibacillus strain, which isselected from the group consisting of:

-   -   1) strain Lu16774 deposited with DSMZ under Accession No. DSM        26969,    -   2) strain Lu17007 deposited with DSMZ under Accession No. DSM        26970, and    -   3) strain Lu17015 deposited with DSMZ under Accession No. DSM        26971,    -   4) strains having at least one of the identifying        characteristics of one of said strains Lu16774, Lu17007 and        Lu17015.

Another embodiment of the invention relates to an isolated microorganismselected from strains:

-   -   1) Paenibacillus strain Lu16774 deposited with DSMZ under        Accession No. DSM 26969,    -   2) Paenibacillus strain Lu17007 deposited with DSMZ under        Accession No. DSM 26970, and    -   3) Paenibacillus strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971;        showing antagonistic activity against at least one plant        pathogen, and being capable of producing at least one        fusaricidin-type compound; or a mutant strain thereof retaining        said capability, i.e. retaining said antagonistic activity        against at least one plant pathogen, and retaining said        capability of producing at least one fusaricidin-type compound.

A further embodiment relates to a microorganism selected from:

-   -   1) Paenibacillus strain Lu16774 deposited with DSMZ under        Accession No. DSM 26969,    -   2) Paenibacillus strain Lu17007 deposited with DSMZ under        Accession No. DSM 26970, and    -   3) Paenibacillus strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971;        or a mutant strain thereof having all the identifying        characteristics of one of said strains.

An identifying characteristic of the deposited Paenibacillus strainsLu16774, Lu17007 and Lu17015 is that they are capable of producing atleast one compound of formula I, preferably selected from compounds 1Aand 1B, in particular producing compounds 1A and 1B, which aremetabolites of the respective strains; and the agriculturally acceptablesalts thereof.

Thus, according to one aspect of the invention, Paenibacillus strains ofthe invention are capable of producing at least one compound of formulaI, more preferably producing compounds 1A or 1B, in particular producingcompounds 1A and 1B; and the agriculturally acceptable salts thereof.

Thus, according to one aspect of the invention, Paenibacillus strains ofthe invention are capable of producing at least one compound of formulaI, more preferably producing compounds 1A or 1B, in particular producingcompounds 1A and 1B; and the agriculturally acceptable salts thereof, ina growth medium comprising at least one source of carbon and one sourceof nitrogen as defined herein.

Thus, according to one aspect of the invention, Paenibacillus strains ofthe invention in a growth medium comprising at least one source ofcarbon and one source of nitrogen as defined herein produce at least onecompound of formula I, more preferably produce compounds 1A or 1B, inparticular produce compounds 1A and 1B; and the agriculturallyacceptable salts thereof.

Another embodiment of the invention relates to an isolated microorganismselected from

-   -   1) Paenibacillus strain Lu16774 deposited with DSMZ under        Accession No. DSM 26969,    -   2) Paenibacillus strain Lu17007 deposited with DSMZ under        Accession No. DSM 26970, and    -   3) Paenibacillus strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971;        showing antagonistic activity against at least one plant        pathogen, and being capable of producing at least one        fusaricidin-type compound of formula I, preferably selected from        compounds 1A and 1B, in particular producing compounds 1A and        1B; or a mutant strain thereof retaining said capability, i.e.        retaining said antagonistic activity against at least one plant        pathogen, and retaining said capability of producing at least        one fusaricidin-type compound of formula I, preferably selected        from compounds 1A and 1B, in particular producing compounds 1A        and 1B.

Another embodiment of the invention relates to an isolated microorganismselected from

-   -   1) Paenibacillus strain Lu16774 deposited with DSMZ under        Accession No. DSM 26969,    -   2) Paenibacillus strain Lu17007 deposited with DSMZ under        Accession No. DSM 26970, and    -   3) Paenibacillus strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971;        showing antagonistic activity against at least one plant        pathogen, and being in a growth medium comprising at least one        source of carbon and one source of nitrogen as defined herein        capable of producing at least one fusaricidin-type compound of        formula I, preferably selected from compounds 1A and 1B, in        particular producing compounds 1A and 1B; or a mutant strain        thereof retaining said capability, i.e. retaining said        antagonistic activity against at least one plant pathogen, and        retaining said capability of producing at least one        fusaricidin-type compound of formula I, preferably selected from        compounds 1A and 1B, in particular producing compounds 1A and        1B.

Another embodiment of the invention relates to an isolated microorganismselected from

-   -   1) Paenibacillus strain Lu16774 deposited with DSMZ under        Accession No. DSM 26969,    -   2) Paenibacillus strain Lu17007 deposited with DSMZ under        Accession No. DSM 26970, and    -   3) Paenibacillus strain Lu17015 deposited with DSMZ under        Accession No. DSM 26971;        showing antagonistic activity against at least one plant        pathogen, and producing at least one fusaricidin-type compound        of formula I, preferably selected from compounds 1A and 1B, in        particular producing compounds 1A and 1B; or a mutant strain        thereof retaining said capability, i.e. retaining said        antagonistic activity against at least one plant pathogen, and        retaining said capability of producing at least one        fusaricidin-type compound of formula I, preferably selected from        compounds 1A and 1B, in particular producing compounds 1A and        1B.

A further identifying characteristic of the deposited Paenibacillusstrains Lu16774, Lu17007 and Lu17015 or a mutant strain thereof is thatthey are capable of producing at least one compound selected from thegroup consisting of fusaricidin A, fusaricidin B, fusaricidin C,fusaricidin D, LI-F06a, LI-F06b and LI-F08b in addition to theircapability of producing at least one compound of formula I, preferablyselected from compounds 1A and 1B, in particular producing compounds 1Aand 1B.

Thus, according to a further aspect of the invention, Paenibacillusstrains of the invention are capable of producing at least onefusaricidin of formula I, preferably selected from compounds 1A and 1B,in particular producing compounds 1A and 1B, as disclosed herein, andare capable of producing at least one compound selected from the groupconsisting of fusaricidin A, fusaricidin B, fusaricidin C, fusaricidinD, LI-F06a, LI-F06b and LI-F08b.

According to a further aspect of the invention, Paenibacillus strains ofthe invention are capable of producing at least one fusaricidin offormula I, preferably selected from compounds 1A and 1B, in particularproducing compounds 1A and 1B, as disclosed herein, and are capable ofproducing at least three compounds selected from the group consisting offusaricidin A, fusaricidin B, fusaricidin C, fusaricidin D, LI-F06a,LI-F06b and LI-F08b.

According to a further aspect of the invention, Paenibacillus strains ofthe invention are capable of producing at least one fusaricidin offormula I, preferably selected from compounds 1A and 1B, in particularproducing compounds 1A and 1B, as disclosed herein, and are capable ofproducing at least five compounds selected from the group consisting offusaricidin A, fusaricidin B, fusaricidin C, fusaricidin D, LI-F06a,LI-F06b and LI-F08b.

According to a further aspect of the invention, Paenibacillus strains ofthe invention are capable of producing at least one fusaricidin offormula I, preferably selected from compounds 1A and 1B, in particularproducing compounds 1A and 1B, as disclosed herein, and are capable ofproducing fusaricidin A, fusaricidin B, fusaricidin C, fusaricidin D andLI-F08b.

A further identifying characteristic of the deposited Paenibacillusstrains are their antifungal activity. In particular, these strains werefound to be effective against infestion with plant pathogens includingAlternaria spp., Botrytis cinerea, Phytophthora infestans, andSclerotinia sclerotiorum; wherein Alternaria spp. is preferably selectedfrom A. solani and A. alternata, in particular A. solani.

Thus, according to a further aspect of the invention, Paenibacillusstrains of the invention have antifungal activity, particularly againsta plant pathogen selected from the group consisting of Alternaria spp.,Botrytis cinerea, Phytophthora infestans, and Sclerotinia sclerotiorum,wherein Alternaria spp. is preferably selected from A. solani and A.alternata, in particular A. solani. More particularly, Paenibacillusstrains of the invention have antifungal activity against at least twoor against all four of said pathogens.

According to a further aspect of the invention, Paenibacillus strains ofthe invention have antifungal activity against the plant pathogensAlternaria solani, Botrytis cinerea, Phytophthora infestans, andSclerotinia sclerotiorum.

Antagonistic activity of the Paenibacillus strains against plantpathogens can be shown in an in-vitro confrontation assays using thedesired phytopathogenic fungi such as Alternaria spp., Botrytis cinerea,Phytophthora infestans, and Sclerotinia sclerotiorum wherein Alternariaspp. is preferably selected from A. solani and A. alternata, inparticular A. solani.

As growth medium for these phytopathogenic fungi, ISP2 medium is usedcomprising per litre: 10 g malt extract (Sigma Aldrich, 70167); 4 gBacto yeast extract (Becton Dickinson, 212750); 4 g glucose monohydrate(Sigma Aldrich, 16301); 20 g Agar (Becton Dickinson, 214510), pH about7, Aq. bidest. As growth medium for PHYTIN, V8 medium is used comprisingper litre: 200 ml of vegetable juice, 3 g calcium carbonate (MerckMillipore, 1020660250); 30 g Agar (Becton Dickinson, 214510), pH 6.8,Aq. bidest.

The Paenibacillus strains are point-inoculated on one side of an agarplate. An agar block (approx. 0.3 cm²) containing one actively growingplant pathogen was put in the center of the plate. After incubating for7-14 days at about 25° C., the growth of the plant pathogen is examined,especially for inhibition zones. The following antagonistic effects canbe evaluated: Antibiosis is scored by evaluation of the diameter of thefungi-free zone (zone of inhibition). Competition is scored by comparingthe diameter of the growth of the fungal pathogen on plates withbacterial strains in comparison to control plates. Mycoparasitism can bedocumented in case the bacteria overgrows the fungal pathogen and alsomycoparasite the pathogens. This can be visualized by microscopy.

Another identifying characteristic of the deposited Paenibacillusstrains Lu16774, Lu17007 and Lu17015 is that they are capable ofproducing and secreting at least one lytic enzyme preferably selectedfrom chitinase, cellulase and amylase (see Example 6), even morepreferably at least chitinase and cellulose; in particular in a growthmedium comprising at least one source of carbon and one source ofnitrogen as defined herein.

Thus, according to a further aspect of the invention, Paenibacillusstrains of the invention are capable of producing and secreting at leastone lytic enzyme preferably selected from chitinase, cellulase andamylase, even more preferably at least chitinase and cellulose; inparticular in a growth medium comprising at least one source of carbonand one source of nitrogen as defined herein.

More specifically, the present invention relates to the depositedstrains Lu16774, Lu17007 and Lu17015 and any Paenibacillus strain havingone or more of the identifying characteristics of the deposited strain,wherein the identifying characteristics are selected from the groupconsisting of:

-   -   (a) antifungal activity against a plant pathogen selected from        the group consisting of Alternaria spp., Botrytis cinerea,        Phytophthora infestans, and Sclerotinia sclerotiorum, wherein        Alternaria spp. is preferably selected from A. solani and A.        alternata, in particular A. solani, as disclosed herein;    -   (b) the capability of producing at least one fusaricidin-type        compound of formula I, in particular compounds 1A and/or 1B, as        disclosed herein;    -   (c) the capability of producing at least one compound selected        from the group consisting of fusaricidins A, B, C, D, LI-F06a,        LI-F06b and LI-F08b, as disclosed herein; and    -   (d) the capability of producing and secreting at least one lytic        enzyme selected from the group consisting of chitinase,        cellulose and amylase, as disclosed herein.        -   More preferably, said Paenibacillus strain has the            capabilities referred to as (b), (c) and (d) in a growth            medium comprising at least one source of carbon and one            source of nitrogen as defined herein.

In particular, Paenibacillus strains of the invention have two or moreof the identifying characteristics of the deposited strain, with strainshaving at least the characteristics (a) and (b) being particularlypreferred. For instance, according to a preferred embodiment, thestrains of the invention (a) have an antifungal activity against a plantpathogen selected from the group consisting of Alternaria spp., Botrytiscinerea, Phytophthora infestans, and Sclerotinia sclerotiorum, whereinAlternaria spp. is preferably selected from A. solani and A. alternata,in particular A. solani and (b) are capable of producing at least onecompound of formula I, and particularly compound 1B. According to afurther preferred embodiment, the strains of the invention (a) have anantifungal activity against three or against all of the plant pathogensselected from the group consisting of Alternaria spp., Botrytis cinerea,Phytophthora infestans; and Sclerotinia sclerotiorum, wherein Alternariaspp. is preferably selected from A. solani and A. alternata, inparticular A. solani and (b) are capable of producing at least onecompound of formula I, more preferably producing compounds 1A or 1B, inparticular of producing compounds 1A and 1B.

According to an embodiment of the invention, the strains of theinvention are provided in isolated or substantially purified form.

The terms “isolated” or “substantially purified” are meant to denotethat the strains of the invention have been removed from a naturalenvironment and have been isolated or separated, and are at least 60%free, preferably at least 75% free, and more preferably at least 90%free, even more preferably at least 95% free, and most preferably atleast 99% free from other components with which they were naturallyassociated. An isolate obtained by culturing a single microbial colonyis an example of an isolated strain of the invention.

The strains of the invention may be provided in any physiological statesuch as active or dormant. Dormant strains may be provided for examplefrozen, dried, or lyophilized or partly desiccated (procedures toproduce partly desiccated organisms are given in WO 2008/002371) or inform of spores.

According to an embodiment of the invention, the strains of theinvention are provided in the form of spores.

According to a further embodiment of the invention, the strains of theinvention are provided as a whole culture broth comprising a strain ofthe invention.

The culture is preferably an isolated or substantially purified culture.

An “isolated culture” or “substantially purified culture” refers to aculture of the strains of the invention that does not includesignificant amounts of other materials which normally are found innatural habitat in which the strain grows and/or from which the strainnormally may be obtained. Consequently, such “isolated culture” or“substantially purified culture” is at least 60% free, preferably atleast 75% free, and more preferably at least 90% free, even morepreferably at least 95% free, and most preferably at least 99% free fromother materials which normally are found in natural habitat in which thestrain grows and/or from which the strain normally may be obtained. Suchan “isolated culture” or “substantially purified culture” does normallynot include any other microorganism in quantities sufficient tointerfere with the replication of the strain of the invention. Isolatedcultures of the invention may, however, be combined to prepare a mixedculture of the strains of the invention and a further biopesticide,preferably a microbial pesticide.

The invention relates to methods for the fermentative production ofantipathogenic biopesticides as described herein.

The strains as used according to the invention can be cultivatedcontinuously or discontinuously in the batch process or in the fed batchor repeated fed batch process. A review of known methods of cultivationwill be found in the textbook by Chmiel (Bioprozesstechnik 1. Einführungin die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) orin the textbook by Storhas (Bioreaktoren and periphere Einrichtungen(Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The medium that is to be used for cultivation of the microorganism mustsatisfy the requirements of the particular strains in an appropriatemanner. Descriptions of culture media for various microorganisms aregiven in the handbook “Manual of Methods for General Bacteriology” ofthe American Society for Bacteriology (Washington D. C., USA, 1981).

These media that can be used according to the invention generallycomprise one or more sources of carbon, sources of nitrogen, inorganicsalts, vitamins and/or trace elements. Preferred sources of carbon aresugars, such as mono-, di- or polysaccharides. Very good sources ofcarbon are for example glucose, fructose, mannose, galactose, ribose,sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch orcellulose. Sugars can also be added to the media via complex compounds,such as molasses, or other by-products from sugar refining. It may alsobe advantageous to add mixtures of various sources of carbon. Otherpossible sources of carbon are oils and fats such as soybean oil,sunflower oil, peanut oil and coconut oil, fatty acids such as palmiticacid, stearic acid or linoleic acid, alcohols such as glycerol, methanolor ethanol and organic acids such as acetic acid or lactic acid. Sourcesof nitrogen are usually organic or inorganic nitrogen compounds ormaterials containing these compounds. Examples of sources of nitrogeninclude ammonia gas or ammonium salts, such as ammonium sulfate,ammonium chloride, ammonium phosphate, ammonium carbonate or ammoniumnitrate, nitrates, urea, amino acids or complex sources of nitrogen,such as corn-steep liquor, soybean flour, soybean protein, yeastextract, meat extract and others. The sources of nitrogen can be usedseparately or as a mixture. Inorganic salt compounds that may be presentin the media comprise the chloride, phosphate or sulfate salts ofcalcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese,zinc, copper and iron. Inorganic sulfur-containing compounds, forexample sulfates, sulfites, dithionites, tetrathionates, thiosulfates,sulfides, but also organic sulfur compounds, such as mercaptans andthiols, can be used as sources of sulfur. Phosphoric acid, potassiumdihydrogenphosphate or dipotassium hydrogenphosphate or thecorresponding sodium-containing salts can be used as sources ofphosphorus. Chelating agents can be added to the medium, in order tokeep the metal ions in solution. Especially suitable chelating agentscomprise dihydroxyphenols, such as catechol or protocatechuate, ororganic acids, such as citric acid. The fermentation media usedaccording to the invention may also contain other growth factors, suchas vitamins or growth promoters, which include for example biotin,riboflavin, thiamine, folic acid, nicotinic acid, pantothenate andpyridoxine. Growth factors and salts often come from complex componentsof the media, such as yeast extract, molasses, corn-steep liquor and thelike. In addition, suitable precursors can be added to the medium. Theprecise composition of the compounds in the medium is strongly dependenton the particular experiment and must be decided individually for eachspecific case. Information on media optimization can be found in thetextbook “Applied Microbiol. Physiology, A Practical Approach” (Publ. P.M. Rhodes, P. F. Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 9635773). Growing media can also be obtained from commercial suppliers, suchas Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.

Preferred growth media that can be used according to the inventioncomprise one or more sources of carbon selected from L-arabinose,N-acetyl-D-glucosamine, D-galactose, L-aspartaic acid, D-trehalose,D-mannose, glycerol, D-gluconic acid, D-xylose, D-mannitol, D-ribose,D-fructose, α-D-glucose, maltose, D-melibiose, thymidine,α-methyl-D-Galactoside, α-D-lactose, lactulose, sucrose, uridine,α-hydroxy glutaric acid-γ-lactone, β-methyl-D-glucoside, adonitol,maltotriose, 2-deoxyadenosine, adenosine, citric acid, mucic acid,D-cellobiose, inosine, L-serine, L-alanyl-glycine, D-galacturonic acid,α-cyclodextrin, 3-cyclodextrin, dextrin, inulin, pectin, amygdalin,gentiobiose, lactitol, D-melezitose, α-methyl-D-glucoside,3-methyl-D-galactoside, β-methyl-D-xyloside, palatinose, D-raffinose,stachyose, turanose, γ-amino butyric acid, D-gluosamine, D-lactic acid,L-lysine, 3-hydroxy 2-butanone; and one or more sources of nitrogenselected from ammonia, nitrite, nitrate, L-alaninie, L-asparagine,L-aspartic acid, L-glutamic acid, L-glutamie, glycine, aminoacid dimes:Ala-Asp, AlaGln, Ala-Glu, Ala-His, Gly-Gln, Gly-Glu, Gly-Met, andMet-Ala; in particular nitrate. These media can be supplemented withinorganic salts and vitamins and/or trace elements. The strains arecapable to produce compounds 1A and 1B in these growth media.

All components of the medium are sterilized, either by heating (20 minat 2.0 bar and 121° C.) or by sterile filtration. The components can besterilized either together, or if necessary separately. All thecomponents of the medium can be present at the start of growing, oroptionally can be added continuously or by batch feed.

The temperature of the culture is normally between 15° C. and 36° C.,preferably 25° C. to 33° C. and can be kept constant or can be variedduring the experiment. The pH value of the medium should be in the rangefrom 5 to 8.5, preferably around 7.0. The pH value for growing can becontrolled during growing by adding basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or ammonia water or acidcompounds such as phosphoric acid or sulfuric acid. Antifoaming agents,e. g. fatty acid polyglycol esters, can be used for controlling foaming.To maintain the stability of plasmids, suitable substances withselective action, e. g. antibiotics, can be added to the medium. Oxygenor oxygen-containing gas mixtures, e. g. the ambient air, are fed intothe culture in order to maintain aerobic conditions. The temperature ofthe culture is normally from 20° C. to 45° C. Culture is continued untila maximum of the desired product has formed. This is normally achievedwithin 10 hours to 160 hours.

In particular, the strains of the invention may be cultivated in amedium a variety of standard microbiology media such as Luria-BertaniBroth (LB), trypticase-soy broth (TSB), yeast extract/maltextract/glucose broth (YMG, ISP2) at 15° C. to 36° C. for 18 to 360 h inliquid media or in agar-solidified media on a petri dish. Aeration maybe necessary. The bacterial cells (vegetative cells and spores) can bewashed and concentrated (e. g. by centrifugation at temperatures ofabout 15 to 30° C. for about 15 min at 7,000×g).

The invention also relates to culture medium obtainable by culturing thestrains of the invention in a medium and separating the medium from theculture broth (thus, remaining when cells grown in the medium areremoved from the whole culture broth), e. g., the supernatant of a wholeculture broth, i.e., the liquid broth remaining when cells grown inbroth and other debris are removed by centrifugation, filtration,sedimentation, or other means well known in the art. The supernatant canbe obtained e. g. by centrifugation at temperatures of about 2 to 30° C.(more preferably at temperatures of 4 to 20° C.) for about 10 to 60 min(more preferably about 15 to 30 min) at about 5,000 to 20,000×g (morepreferably at about 15,000×g).

Such culture medium contains pesticidal metabolites which are producedby the cultured strain.

The invention also relates to cell-free extracts of the strains of theinvention. To produce a cell-free extract, the strains of the inventionmay be cultivated as described above. The cells can be disrupted also byhigh-frequency ultrasound, by high pressure, e. g. in a French pressurecell, by osmolysis, by the action of detergents, lytic enzymes ororganic solvents, by means of homogenizers or by a combination ofseveral of the methods listed. The extraction can be carried outpreferably with an organic solvent or solvent mixture, more preferablyan alcohol (e. g. methanol, ethanol, n-propanol, 2-propanol or alike),even more preferably with 2-propanol (e. g. in a 1:1 ratio to theculture volume). Phase separation may be enhanced by addition of saltssuch as NaCl. The organic phase can be collected and the solvent orsolvent mixture may be removed by conventional distillation and/ordrying followed by resuspension in methanol and filtration.

Such extract contains pesticidal metabolites which are produced by thecultured strain.

Pesticidal metabolites that are specific to the strains of the inventionmay be recovered from such medium or extract according to conventionalmethods in particular when the strains of the invention have beencultivated as described above.

The methodology of the present invention can further include a step ofrecovering individual pesticidal metabolites.

The term “recovering” includes extracting, harvesting, isolating orpurifying the compound from culture media or cell-free extracts.Recovering the compound can be performed according to any conventionalisolation or purification methodology known in the art including, butnot limited to, treatment with a conventional resin (e. g., anion orcation exchange resin, non-ionic adsorption resin, etc.), treatment witha conventional adsorbent (e. g., activated charcoal, silicic acid,silica gel, cellulose, alumina, etc.), alteration of pH, solventextraction (e. g., with a conventional solvent such as an alcohol, ethylacetate, hexane and the like), distillation, dialysis, filtration,concentration, crystallization, recrystallization, pH adjustment,lyophilization and the like. For example the metabolites can berecovered from culture media by first removing the microorganisms. Theremaining broth is then passed through or over a cation exchange resinto remove unwanted cations and then through or over an anion exchangeresin to remove unwanted inorganic anions and organic acids.

Several metabolites have been found in whole culture broth of the novelPaenibacillus strains. Nine metabolites have been studied in detail andidentified (see Example 7, FIG. 1). Two of them were found to be novel(compound 1A and compound 1B). Compounds 1A and 1B have been found to beproduced by all three Paenibacillus strains of the invention (see Table17) but none of them was found in the whole culture broth of the relatedPaenibacillus peoriae strain NRRL BD-62.

Thus the present invention also relates to compounds of formula I

wherein

-   R is selected from 15-guanidino-3-hydroxypentadecanoic acid (GHPD)    and 12-guanidinododecanoic acid (12-GDA);-   X¹ is threonine;-   X² is isoleucine;-   X³ is tyrosine;-   X⁴ is threonine;-   X⁵ is selected from glutamine and asparagine;-   X⁶ is alanine; and    wherein an arrow defines a single (amide) bond either between the    carbonyl moiety of R and the amino group of the amino acid X¹ or    between the carbonyl group of one amino acid and the amino group of    a neighboring amino acid wherein the tip of the arrow indicates the    attachment to the amino group of said neighboring amino acid; and    wherein the single line (without an arrow head) defines a single    (ester) bond between the carbonyl group of X⁶ and the hydroxyl group    of X¹;    and the agriculturally acceptable salts thereof.

According to a further embodiment, X¹ in formula I is preferablyL-threonine.

According to a further embodiment, X² in formula I is preferablyD-isoleucine or D-allo-isoleucine.

According to a further embodiment, X³ in formula I is preferablyL-tyrosine.

According to a further embodiment, X⁴ in formula I is preferablyD-allo-threonine.

According to a further embodiment, X⁵ in formula I is preferablyD-glutamine or D-asparagine.

According to a further embodiment, R in formula I is preferably GHPD.

The sketch of formula I for compounds of formula I may also be depictedas follows:

wherein

-   X is selected from —NH—(C═O)—CH₂—CH(OH)—(CH₂)₁₂—NH—C(═NH)NH₂ and    —NH—(C═O)—(CH₂)₁₁—NH—C(═NH)NH₂;-   R¹ is 1-hydroxyethyl;-   R² is 1-methylpropyl (sec-butyl);-   R³ is 4-hydroxybenzyl;-   R⁴ is 1-hydroxyethyl;-   R⁵ is selected from carbamoylethyl and carbamoylmethyl;-   R⁶ is methyl.

Likewise, the preferred embodiments based on this alternative sketch offormula I

are as follows:

R¹ in this formula I is preferably (1S,2R)-1-hydroxyethyl.

R² in this formula I is preferably (1R,2R)-1-methylpropyl or(1R,2S)-1-methylpropyl.

R³ in this formula I is preferably (S)-4-hydroxy-benzyl.

R⁴ in this formula I is preferably (1S,2R)-1-hydroxyethyl.

R⁵ in this formula I is preferably (R)-carbamoylethyl and(R)-carbamoylmethyl.

X in this formula I is preferably—NH—(C═O)—CH₂—CH(OH)—(CH₂)₁₂—NH—C(═NH)NH₂.

According to a further embodiment, the invention further relates tocompounds 1A and 1B, which are of formula I, wherein R is GHPD andwherein X⁴ is asparagine in case of compound 1A and X⁴ is glutamine incase of compound 1B:

The pesticidal metabolites from the strains of the invention arepreferably selected from compounds of formula I wherein R is GHPD, inparticular selected from compounds 1A and 1B, which can be obtained byextraction and isolation from cultures, i.e. whole culture broths, ofthe strains of the invention.

Further, the fusaricidin-type compounds of formula I including thosewherein R is GHTD can be synthesized in analogy to methods known in theart (Biopolymers 80(4), 541, 2005; J. Peptide Sci. 125, 219, 2006;Tetrahedron Lett. 47(48), 8587-90, 2006; Biopolymers 88(4), 568, 2007;ChemMedChem 7, 871-882, 2012).

The invention also relates to the agriculturally acceptable salts,particularly acid addition salts of said fusaricidin-type compounds offormula I. Said salts can be obtained by conventional methods well knownin the art, e. g. by reacting the compounds of the invention with asuitable acid to form an acid addition salt. Anions of useful acidaddition salts are primarily chloride, bromide, fluoride, iodide,hydrogensulfate, methylsulfate, sulfate, dihydrogenphosphate,hydrogen-phosphate, nitrate, bicarbonate, carbonate, hexafluorosilicate,hexafluorophosphate, benzoate and also the anions of C₁-C₄-alkanoicacids, preferably formate, acetate, propionate and butyrate.

Consequently, the invention also relates to a whole culture broth of amicroorganism comprising at least one compound of formula I or anagriculturally acceptable salt thereof, preferably selected fromcompounds 1A and 1B or an agriculturally acceptable salt thereof, inparticular said whole culture broth comprises compounds 1A and 1B or anagriculturally acceptable salt thereof.

According to a further embodiment, the invention also relates to a wholeculture broth of a microorganism of the genus Paenibacillus comprisingat least one compound of formula I or an agriculturally acceptable saltthereof, preferably selected from compounds 1A and 1B or anagriculturally acceptable salt thereof, in particular said whole culturebroth comprises compounds 1A and 1B or an agriculturally acceptable saltthereof.

According to a further embodiment, the invention also relates to a wholeculture broth of at least one Paenibacillus strain of the invention asidentified and defined above comprising at least one compound of formulaI or an agriculturally acceptable salt thereof, preferably selected fromcompounds 1A and 1B or an agriculturally acceptable salt thereof, inparticular said whole culture broth comprises compounds 1A and 1B or anagriculturally acceptable salt thereof.

Said fusaricidin-type compounds are secreted into the culture medium ofthe respective microorganism capable of producing it.

Consequently, the invention also relates to a culture medium and/or acell-free extract of a microorganism comprising at least one compound offormula I or an agriculturally acceptable salt thereof, preferablyselected from compounds 1A and 1B or an agriculturally acceptable saltthereof, in particular said culture medium and/or a cell-free extractcomprises compounds 1A and 1B or an agriculturally acceptable saltthereof.

According to a further embodiment, the invention also relates to aculture medium and/or a cell-free extract of a microorganism of thegenus Paenibacillus comprising at least one compound of formula I or anagriculturally acceptable salt thereof, preferably selected fromcompounds 1A and 1B or an agriculturally acceptable salt thereof, inparticular said culture medium and/or a cell-free extract comprisescompounds 1A and 1B or an agriculturally acceptable salt thereof.

According to a further embodiment, the invention also relates to culturemedium and/or a cell-free extract of at least one Paenibacillus strainof the invention as identified and defined above comprising at least onecompound of formula I or an agriculturally acceptable salt thereof,preferably selected from compounds 1A and 1B or an agriculturallyacceptable salt thereof, in particular said culture medium and/or acell-free extract comprises compounds 1A and 1B or an agriculturallyacceptable salt thereof.

The invention further relates to agrochemical compositions comprising anauxiliary as defined below and at least one or more of the strains,whole culture broths, cell-free extracts, culture media and compounds offormula I, of the invention, respectively.

As used herein, “composition” in reference to a product (microbialstrain, agent or formulation) of the present invention refers to acombination of ingredients, wherein “formulating” is the process ofusing a formula, such as a recipe, for a combination of ingredients, tobe added to form the formulation. Such composition is also referredherein to as formulation.

The strains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively,are suitable as antifungal agents or fungicides. They are distinguishedby an outstanding effectiveness against a broad spectrum ofphytopathogenic fungi, including soil-borne fungi, which deriveespecially from the classes of the Plasmodiophoromycetes,Peronosporomycetes (syn. Oomycetes), Chytridiomycetes, Zygomycetes,Ascomycetes, Basidiomycetes and Deuteromycetes (syn. Fungi imperfecti).Some are systemically effective and they can be used in crop protectionas foliar fungicides, fungicides for seed dressing and soil fungicides.Moreover, they are suitable for controlling harmful fungi, which interalia occur in wood or roots of plants.

The strains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively,are particularly important in the control of a multitude ofphytopathogenic fungi on various cultivated plants, such as cereals, e.g. wheat, rye, barley, triticale, oats or rice; beet, e. g. sugar beetor fodder beet; fruits, such as pomes, stone fruits or soft fruits, e.g. apples, pears, plums, peaches, almonds, cherries, strawberries,raspberries, blackberries or gooseberries; leguminous plants, such aslentils, peas, alfalfa or soybeans; oil plants, such as rape, mustard,olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms,ground nuts or soybeans; cucurbits, such as squashes, cucumber ormelons; fiber plants, such as cotton, flax, hemp or jute; citrus fruit,such as oranges, lemons, grapefruits or mandarins; vegetables, such asspinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes,potatoes, cucurbits or paprika; lauraceous plants, such as avocados,cinnamon or camphor; energy and raw material plants, such as corn,soybean, rape, sugar cane or oil palm; corn; tobacco; nuts; coffee; tea;bananas; vines (table grapes and grape juice grape vines); hop; turf;sweet leaf (also called Stevia); natural rubber plants or ornamental andforestry plants, such as flowers, shrubs, broad-leaved trees orevergreens, e. g. conifers; and on the plant propagation material, suchas seeds, and the crop material of these plants.

Preferably, the strains, whole culture broths, cell-free extractsculture media, compounds of formula I; and compositions of theinvention, respectively, are used for controlling a multitude of fungion field crops, such as potatoes sugar beets, tobacco, wheat, rye,barley, oats, rice, corn, cotton, soybeans, rape, legumes, sunflowers,coffee or sugar cane; fruits; vines; ornamentals; or vegetables, such ascucumbers, tomatoes, beans or squashes.

The term “plant propagation material” is to be understood to denote allthe generative parts of the plant such as seeds and vegetative plantmaterial such as cuttings and tubers (e. g. potatoes), which can be usedfor the multiplication of the plant. This includes seeds, roots, fruits,tubers, bulbs, rhizomes, shoots, sprouts and other parts of plants,including seedlings and young plants, which are to be transplanted aftergermination or after emergence from soil. These young plants may also beprotected before transplantation by a total or partial treatment byimmersion or pouring.

Preferably, treatment of plant propagation materials with the strains,whole culture broths, cell-free extracts culture media, compounds offormula I; and compositions of the invention, respectively, is used forcontrolling a multitude of fungi on cereals, such as wheat, rye, barleyand oats; rice, corn, cotton and soybeans.

The term “cultivated plants” is to be understood as including plantswhich have been modified by breeding, mutagenesis or genetic engineeringincluding but not limiting to agricultural biotech products on themarket or in development (cf. http://cera-gmc.org/, see GM crop databasetherein). Genetically modified plants are plants, which genetic materialhas been so modified by the use of recombinant DNA techniques that undernatural circumstances cannot readily be obtained by cross breeding,mutations or natural recombination. Typically, one or more genes havebeen integrated into the genetic material of a genetically modifiedplant in order to improve certain properties of the plant. Such geneticmodifications also include but are not limited to targetedpost-translational modification of protein(s), oligo- or polypeptides e.g. by glycosylation or polymer additions such as prenylated, acetylatedor farnesylated moieties or PEG moieties.

Plants that have been modified by breeding, mutagenesis or geneticengineering, e. g. have been rendered tolerant to applications ofspecific classes of herbicides, such as auxin herbicides such as dicambaor 2,4-D; bleacher herbicides such as hydroxylphenylpyruvate dioxygenase(HPPD) inhibitors or phytoene desaturase (PDS) inhibitors; acetolactatesynthase (ALS) inhibitors such as sulfonyl ureas or imidazolinones;enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, such asglyphosate; glutamine synthetase (GS) inhibitors such as glufosinate;protoporphyrinogen-IX oxidase inhibitors; lipid biosynthesis inhibitorssuch as acetyl CoA carboxylase (ACCase) inhibitors; or oxynil (i. e.bromoxynil or ioxynil) herbicides as a result of conventional methods ofbreeding or genetic engineering. Furthermore, plants have been maderesistant to multiple classes of herbicides through multiple geneticmodifications, such as resistance to both glyphosate and glufosinate orto both glyphosate and a herbicide from another class such as ALSinhibitors, HPPD inhibitors, auxin herbicides, or ACCase inhibitors.These herbicide resistance technologies are e. g. described in PestManagem. Sci. 61, 2005, 246; 61, 2005, 258; 61, 2005, 277; 61, 2005,269; 61, 2005, 286; 64, 2008, 326; 64, 2008, 332; Weed Sci. 57, 2009,108; Austral. J. Agricult. Res. 58, 2007, 708; Science 316, 2007, 1185;and references quoted therein. Several cultivated plants have beenrendered tolerant to herbicides by conventional methods of breeding(mutagenesis), e. g. Clearfield® summer rape (Canola, BASF SE, Germany)being tolerant to imidazolinones, e. g. imazamox, or ExpressSun®sunflowers (DuPont, USA) being tolerant to sulfonyl ureas, e. g.tribenuron. Genetic engineering methods have been used to rendercultivated plants such as soybean, cotton, corn, beets and rape,tolerant to herbicides such as glyphosate and glufosinate, some of whichare commercially available under the trade names RoundupReady®(glyphosate-tolerant, Monsanto, U.S.A.), Cultivance® (imidazolinonetolerant, BASF SE, Germany) and LibertyLink® (glufosinate-tolerant,Bayer CropScience, Germany).

Furthermore, plants are also covered that are by the use of recombinantDNA techniques capable to synthesize one or more insecticidal proteins,especially those known from the bacterial genus Bacillus, particularlyfrom Bacillus thuringiensis, such as 6-endotoxins, e. g. CryIA(b),CryIA(c), CryIF, CryIF(a2), CryIIA(b), CryIIIA, CryIIIB(b1) or Cry9c;vegetative insecticidal proteins (VIP), e. g. VIP1, VIP2, VIP3 or VIP3A;insecticidal proteins of bacteria colonizing nematodes, e. g.Photorhabdus spp. or Xenorhabdus spp.; toxins produced by animals, suchas scorpion toxins, arachnid toxins, wasp toxins, or otherinsect-specific neurotoxins; toxins produced by fungi, suchStreptomycetes toxins, plant lectins, such as pea or barley lectins;agglutinins; proteinase inhibitors, such as trypsin inhibitors, serineprotease inhibitors, patatin, cystatin or papain inhibitors;ribosome-inactivating proteins (RIP), such as ricin, maize-RIP, abrin,luffin, saporin or bryodin; steroid metabolism enzymes, such as3-hydroxysteroid oxidase, ecdysteroid-IDP-glycosyltransferase,cholesterol oxidases, ecdysone inhibitors or HMG-CoA-reductase; ionchannel blockers, such as blockers of sodium or calcium channels;juvenile hormone esterase; diuretic hormone receptors (helicokininreceptors); stilbene synthase, bibenzyl synthase, chitinases orglucanases. In the context of the present invention these insecticidalproteins or toxins are to be understood expressly also as pre-toxins,hybrid proteins, truncated or otherwise modified proteins. Hybridproteins are characterized by a new combination of protein domains,(see, e. g. WO 02/015701). Further examples of such toxins orgenetically modified plants capable of synthesizing such toxins aredisclosed, e. g., in EP-A 374 753, WO 93/007278, WO 95/34656, EP-A 427529, EP-A 451 878, WO 03/18810 and WO 03/52073. The methods forproducing such genetically modified plants are generally known to theperson skilled in the art and are described, e. g. in the publicationsmentioned above. These insecticidal proteins contained in thegenetically modified plants impart to the plants producing theseproteins tolerance to harmful pests from all taxonomic groups ofarthropods, especially to beetles (Coeloptera), two-winged insects(Diptera), and moths (Lepidoptera) and to nematodes (Nematoda).Genetically modified plants capable to synthesize one or moreinsecticidal proteins are, e. g., described in the publicationsmentioned above, and some of which are commercially available such asYieldGard® (corn cultivars producing the Cry1Ab toxin), YieldGard® Plus(corn cultivars producing Cry1Ab and Cry3Bb1 toxins), Starlink® (corncultivars producing the Cry9c toxin), Herculex® RW (corn cultivarsproducing Cry34Ab1, Cry35Ab1 and the enzymePhosphinothricin-N-Acetyltransferase [PAT]); NuCOTN® 33B (cottoncultivars producing the Cry1Ac toxin), Bollgard® I (cotton cultivarsproducing the Cry1Ac toxin), Bollgard® II (cotton cultivars producingCry1Ac and Cry2Ab2 toxins); VIPCOT® (cotton cultivars producing aVIP-toxin); NewLeaf® (potato cultivars producing the Cry3A toxin);Bt-Xtra®, NatureGard®, KnockOut®, BiteGard®, Protecta®, Bt11 (e. g.Agrisure® CB) and Bt176 from Syngenta Seeds SAS, France, (corn cultivarsproducing the CrylAb toxin and PAT enzyme), MIR604 from Syngenta SeedsSAS, France (corn cultivars producing a modified version of the Cry3Atoxin, c.f. WO 03/018810), MON 863 from Monsanto Europe S.A., Belgium(corn cultivars producing the Cry3Bb1 toxin), IPC 531 from MonsantoEurope S.A., Belgium (cotton cultivars producing a modified version ofthe Cry1Ac toxin) and 1507 from Pioneer Overseas Corporation, Belgium(corn cultivars producing the Cry1F toxin and PAT enzyme).

Furthermore, plants are also covered that are by the use of recombinantDNA techniques capable to synthesize one or more proteins to increasethe resistance or tolerance of those plants to bacterial, viral orfungal pathogens. Examples of such proteins are the so-called“pathogenesis-related proteins” (PR proteins, see, e. g. EP-A 392 225),plant disease resistance genes (e. g. potato cultivars, which expressresistance genes acting against Phytophthora infestans derived from themexican wild potato Solanum bulbocastanum) or T4-lysozym (e. g. potatocultivars capable of synthesizing these proteins with increasedresistance against bacteria such as Erwinia amylvora). The methods forproducing such genetically modified plants are generally known to theperson skilled in the art and are described, e. g. in the publicationsmentioned above.

Furthermore, plants are also covered that are by the use of recombinantDNA techniques capable to synthesize one or more proteins to increasethe productivity (e. g. bio mass production, grain yield, starchcontent, oil content or protein content), tolerance to drought, salinityor other growth-limiting environmental factors or tolerance to pests andfungal, bacterial or viral pathogens of those plants.

Furthermore, plants are also covered that contain by the use ofrecombinant DNA techniques a modified amount of substances of content ornew substances of content, specifically to improve human or animalnutrition, e. g. oil crops that produce health-promoting long-chainomega-3 fatty acids or unsaturated omega-9 fatty acids (e. g. Nexera®rape, DOW Agro Sciences, Canada).

Furthermore, plants are also covered that contain by the use ofrecombinant DNA techniques a modified amount of substances of content ornew substances of content, specifically to improve raw materialproduction, e. g. potatoes that produce increased amounts of amylopectin(e. g. Amflora® potato, BASF SE, Germany).

The strains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively,are particularly suitable for controlling the following plant diseases:

Albugo spp. (white rust) on ornamentals, vegetables (e. g. A. candida)and sunflowers (e. g. A. tragopogonis); Alternaria spp. (Alternaria leafspot) on vegetables, rape (A. brassicola or brassicae), sugar beets (A.tenuis), fruits, rice, soybeans, potatoes (e. g. A. solani or A.altemata), tomatoes (e. g. A. solani or A. altemata) and wheat;Aphanomycesspp. on sugar beets and vegetables; Ascochyta spp. on cerealsand vegetables, e. g. A. tritici (anthracnose) on wheat and A. hordei onbarley; Bipolaris and Drechslera spp. (teleomorph: Cochliobolus spp.),e. g. Southern leaf blight (D. maydis) or Northern leaf blight (B.zeicola) on corn, e. g. spot blotch (B. sorokiniana) on cereals and e.g. B. oryzae on rice and turfs; Blumeria (formerly Erysiphe) graminis(powdery mildew) on cereals (e. g. on wheat or barley); Botrytis cinerea(teleomorph: Botryotinia fuckeliana. grey mold) on fruits and berries(e. g. strawberries), vegetables (e. g. lettuce, carrots, celery andcabbages), rape, flowers, vines, forestry plants and wheat; Bremialactucae (downy mildew) on lettuce; Ceratocystis (syn. Ophiostoma) spp.(rot or wilt) on broad-leaved trees and evergreens, e. g. C. ulmi (Dutchelm disease) on elms; Cercospora spp. (Cercospora leaf spots) on corn(e. g. Gray leaf spot: C. zeae-maydis), rice, sugar beets (e. g. C.beticola), sugar cane, vegetables, coffee, soybeans (e. g. C. sojina orC. kikuchii) and rice; Cladosporium spp. on tomatoes (e. g. C. fulvumleaf mold) and cereals, e. g. C. herbarum (black ear) on wheat;Claviceps purpurea (ergot) on cereals; Cochliobolus (anamorph:Helminthosporium of Bipolaris) spp. (leaf spots) on corn (C. carbonum),cereals (e. g. C. sativus, anamorph: B. sorokiniana) and rice (e. g. C.miyabeanus, anamorph: H. oryzae); Colletotrichum (teleomorph:Glomerella) spp. (anthracnose) on cotton (e. g. C. gossypii), corn (e.g. C. graminicola: Anthracnose stalk rot), soft fruits, potatoes (e. g.C. coccodes. black dot), beans (e. g. C. lindemuthianum) and soybeans(e. g. C. truncatum or C. gloeosporiodes); Corticium spp., e. g. C.sasakii (sheath blight) on rice; Corynespora cassikola (leaf spots) onsoybeans and ornamentals; Cycloconium spp., e. g. C. oleaginum on olivetrees; Cylindrocarpon spp. (e. g. fruit tree canker or young vinedecline, teleomorph: Nectria or Neonectria spp.) on fruit trees, vines(e. g. C. liriodendri; teleomorph: Neonectria liriodendri: Black FootDisease) and ornamentals; Dematophora (teleomorph: Rosellinla) necatrix(root and stem rot) on soybeans; Diaporthe spp., e. g. D. phaseolorum(damping off) on soybeans; Drechslera (syn. Helminthosporium,teleomorph: Pyrenophora) spp. on corn, cereals, such as barley (e. g. D.teres, net blotch) and wheat (e. g. D. tritici-repentis tan spot), riceand turf; Esca (dieback, apoplexy) on vines, caused by Formitiporia(syn. Phellinus) punctata, F. mediterranea, Phaeomoniella chlamydospora(earlier Phaeoacremonium chlamydosporum), Phaeoacremonium aleophilumand/or Botryosphaeria obtusa; Elsinoe spp. on pome fruits (E. pyri),soft fruits (E. veneta: anthracnose) and vines (E. ampelina:anthracnose); Entyloma oryzae (leaf smut) on rice; Epicoccum spp. (blackmold) on wheat; Erysihe spp. (powdery mildew) on sugar beets (E. betae),vegetables (e. g. E. pisi) such as cucurbits (e. g. E. cichoracearum),cabbages, rape (e. g. E. cruciferarum); Eutypa lata (Eutypa canker ordieback, anamorph: Cytosporina lata, syn. Libertella blepharis) on fruittrees, vines and ornamental woods; Exserohilum (syn. Helminthosporium)spp. on corn (e. g. E. turicum); Fusarium (teleomorph: Gibberella) spp.(wilt, root or stem rot) on various plants, such as F. graminearum or F.culmorum (root rot, scab or head blight) on cereals (e. g. wheat orbarley), F. oxysporum on tomatoes, F. solani (f. sp. glycines now syn.F. virguliforme) and F. tucumaniae and F. brasiliense each causingsudden death syndrome on soybeans, and F. verticillioides on corn;Gaeumannomyces graminis (take-all) on cereals (e. g. wheat or barley)and corn; Gibberella spp. on cereals (e. g. G. zeae) and rice (e. g. G.fufikuroi. Bakanae disease); Glomerella cingulata on vines, pome fruitsand other plants and G. gossypii on cotton; Grainstaining complex onrice; Guignardia bidwellii (black rot) on vines; Gymnosporangium spp. onrosaceous plants and junipers, e. g. G. sabinae (rust) on pears;Helminthosporium spp. (syn. Drechslera, teleomorph: Cochllobolus) oncorn, cereals and rice; Hemileia spp., e. g. H. vastatfix (coffee leafrust) on coffee; Isarlopsis clavispora (syn. Cladosporium vitis) onvines; Macrophomina phaseolina (syn. phaseoli) (root and stem rot) onsoybeans and cotton; Microdochium (syn. Fusarium) nivale (pink snowmold) on cereals (e. g. wheat or barley); Microsphaera diffusa (powderymildew) on soybeans; Monilinia spp., e. g. M. laxa, M. fructicola and M.fructigena (bloom and twig blight, brown rot) on stone fruits and otherrosaceous plants; Mycosphaerella spp. on cereals, bananas, soft fruitsand ground nuts, such as e. g. M. graminicola (anamorph: Septoriatritici, Septoria blotch) on wheat or M. fijensis (black Sigatokadisease) on bananas; Peronospora spp. (downy mildew) on cabbage (e. g.P. brassicae), rape (e. g. P. parasitica), onions (e. g. P. destructor),tobacco (P. tabacina) and soybeans (e. g. P. manshurka); Phakopsorapachyrhizi and P. meibomiae (soybean rust) on soybeans; Phialophora spp.e. g. on vines (e. g. P. tracheiphila and P. tetraspora) and soybeans(e. g. P. gregata stem rot); Phoma lingam (root and stem rot) on rapeand cabbage and P. betae (root rot, leaf spot and damping-off) on sugarbeets; Phomopsis spp. on sunflowers, vines (e. g. P. viticola: can andleaf spot) and soybeans (e. g. stem rot: P. phaseoli teleomorph:Diaporthe phaseolorum); Physoderma maydis (brown spots) on corn;Phytophthora spp. (wilt, root, leaf, fruit and stem root) on variousplants, such as paprika and cucurbits (e. g. P. capsici), soybeans (e.g. P. megasperma, syn. P. sojae), potatoes and tomatoes (e. g. P.infestans late blight) and broad-leaved trees (e. g. P. ramorum suddenoak death); Plasmodiophora brassicae (club root) on cabbage, rape,radish and other plants; Plasmopara spp., e. g. P. viticola (grapevinedowny mildew) on vines and P. halstedii on sunflowers; Podosphaera spp.(powdery mildew) on rosaceous plants, hop, pome and soft fruits, e. g.P. leucotricha on apples; Polymyxa spp., e. g. on cereals, such asbarley and wheat (P. graminis) and sugar beets (P. betae) and therebytransmitted viral diseases; Pseudocercosporella herpotrkhoides (eyespot,teleomorph: Tapesia yallundae) on cereals, e. g. wheat or barley;Pseudoperonospora (downy mildew) on various plants, e. g. P. cubensis oncucurbits or P. humili on hop; Pseudopezicula tracheiphila (red firedisease or ‘rotbrenner’, anamorph: Phialophora) on vines; Puccinia spp.(rusts) on various plants, e. g. P. tritkina (brown or leaf rust), P.striiformis (stripe or yellow rust), P. hordei (dwarf rust), P. graminis(stem or black rust) or P. recondita (brown or leaf rust) on cereals,such as e. g. wheat, barley or rye, P. kuehnii (orange rust) on sugarcane and P. asparagi on asparagus; Pyrenophora (anamorph: Drechslera)tritici-repentis (tan spot) on wheat or P. teres (net blotch) on barley;Pyricularia spp., e. g. P. oryzae (teleomorph: Magnaporthe grisea, riceblast) on rice and P. grisea on turf and cereals; Pythium spp.(damping-off) on turf, rice, corn, wheat, cotton, rape, sunflowers,soybeans, sugar beets, vegetables and various other plants (e. g. P.ultimum or P. aphanidermatum); Ramularia spp., e. g. R. colo-cygni(Ramularia leaf spots, Physiological leaf spots) on barley and R.beticola on sugar beets; Rhizoctonia spp. on cotton, rice, potatoes,turf, corn, rape, potatoes, sugar beets, vegetables and various otherplants, e. g. R. solani (root and stem rot) on soybeans, R. solani(sheath blight) on rice or R. cerealis (Rhizoctonia spring blight) onwheat or barley; Rhizopus stolonifer (black mold, soft rot) onstrawberries, carrots, cabbage, vines and tomatoes; Rhynchosporiumsecalis (scald) on barley, rye and triticale; Sarocladium oryzae and S.attenuatum (sheath rot) on rice; Sclerotinia spp. (stem rot or whitemold) on vegetables and field crops, such as rape, sunflowers (e. g. S.sclerotiorum) and soybeans (e. g. S. rolfsii or S. sclerotiorum);Septoria spp. on various plants, e. g. S. glycines (brown spot) onsoybeans, S. tritici(Septoria blotch) on wheat and S. (syn.Stagonospora) nodorum (Stagonospora blotch) on cereals; Uncinula (syn.Erysiphe) necator (powdery mildew, anamorph: Odium tuckers) on vines;Setospaeria spp. (leaf blight) on corn (e. g. S. turcicum, syn.Helminthosporium turcicum) and turf; Sphacelotheca spp. (smut) on corn,(e. g. S. relliana; head smut), sorghum and sugar cane; Sphaerothecafuliginea (powdery mildew) on cucurbits; Spongospora subterranea(powdery scab) on potatoes and thereby transmitted viral diseases;Stagonospora spp. on cereals, e. g. S. nodorum (Stagonospora blotch,teleomorph: Leptosphaeria [syn. Phaeosphaeria] nodorum) on wheat;Synchytrium endobioticum on potatoes (potato wart disease); Taphrinaspp., e. g. T. deformans (leaf curl disease) on peaches and T. pruni(plum pocket) on plums; Thielaviopsis spp. (black root rot) on tobacco,pome fruits, vegetables, soybeans and cotton, e. g. T. basicola (syn.Chalara elegans); spp. (common bunt or stinking smut) on cereals, suchas e. g. T. tritici (syn. T. caries, wheat bunt) and T. controversa(dwarf bunt) on wheat; Typhula incarnata (grey snow mold) on barley orwheat; Urocystis spp., e. g. U. occulta (stem smut) on rye; Uromycesspp. (rust) on vegetables, such as beans (e. g. U. appendiculatus, syn.U. phaseoli) and sugar beets (e. g. U. betae); Ustilago spp. (loosesmut) on cereals (e. g. U. nuda and U. avaenae), corn (e. g. U. maydiscorn smut) and sugar cane; Venturia spp. (scab) on apples (e. g. V.inaequalis) and pears; and Vertialum spp. (wilt) on various plants, suchas fruits and ornamentals, vines, soft fruits, vegetables and fieldcrops, e. g. V. dahliae on strawberries, rape, potatoes and tomatoes.

The strains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively,are also suitable for controlling harmful pathogens, especially fungi,in the protection of stored products or harvest and in the protection ofmaterials.

The term “protection of materials” is to be understood to denote theprotection of technical and non-living materials, such as adhesives,glues, wood, paper and paperboard, textiles, leather, paint dispersions,plastics, cooling lubricants, fiber or fabrics, against the infestationand destruction by harmful microorganisms, such as fungi and bacteria.As to the protection of wood and other materials, the particularattention is paid to the following harmful fungi: Ascomycetes such asOphiostoma spp., Ceratocystis spp., Aureobasidium pullulans, Sclerophomaspp., Chaetomium spp., Humicola spp., Petriella spp., Trichurus spp.;Basidiomycetes such as Coniophora spp., Coriolus spp., Gloeophyllumspp., Lentinus spp., Pleurotus spp., Poria spp., Serpula spp. andTyromyces spp., Deuteromycetes such as Aspergillus spp., Cladosporiumspp., Penicillium spp., Trichorma spp., Alternaria spp., Paecilomycesspp. and Zygomycetes such as Mucor spp., and in addition in theprotection of stored products and harvest the following yeast fungi areworthy of note: Candida spp. and Saccharomyces cerevisiae.

The method of treatment according to the invention can also be used inthe field of protecting stored products or harvest against attack offungi and microorganisms. According to the present invention, the term“stored products” is understood to denote natural substances of plant oranimal origin and their processed forms, which have been taken from thenatural life cycle and for which long-term protection is desired. Storedproducts of crop plant origin, such as plants or parts thereof, forexample stalks, leafs, tubers, seeds, fruits or grains, can be protectedin the freshly harvested state or in processed form, such as pre-dried,moistened, comminuted, ground, pressed or roasted, which process is alsoknown as post-harvest treatment. Also falling under the definition ofstored products is timber, whether in the form of crude timber, such asconstruction timber, electricity pylons and barriers, or in the form offinished articles, such as furniture or objects made from wood. Storedproducts of animal origin are hides, leather, furs, hairs and the like.The combinations according the present invention can preventdisadvantageous effects such as decay, discoloration or mold. Preferably“stored products” is understood to denote natural substances of plantorigin and their processed forms, more preferably fruits and theirprocessed forms, such as pomes, stone fruits, soft fruits and citrusfruits and their processed forms.

The strains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively,may be used for improving the health of a plant. The invention alsorelates to a method for improving plant health by treating a plant, itspropagation material and/or the locus where the plant is growing or isto grow with an effective amount of the strains, whole culture broths,cell-free extracts, culture media, compounds of formula I, andcompositions.

The term “plant health” is to be understood to denote a condition of theplant and/or its products which is determined by several indicatorsalone or in combination with each other such as yield (e. g. increasedbiomass and/or increased content of valuable ingredients), plant vigor(e. g. improved plant growth and/or greener leaves (“greening effect”)),quality (e. g. improved content or composition of certain ingredients)and tolerance to abiotic and/or biotic stress. The above identifiedindicators for the health condition of a plant may be interdependent ormay result from each other.

Healthier plants are desirable since they result among others in betteryields and/or a better quality of the plants or crops, specificallybetter quality of the harvested plant parts. Healthier plants alsobetter resist to biotic and/or abiotic stress. A high resistance againstbiotic stresses in turn allows the person skilled in the art to reducethe quantity of pesticides applied and consequently to slow down thedevelopment of resistances against the respective pesticides.

It has to be emphasized that the above mentioned effects of the strains,whole culture broths, cell-free extracts, culture media, compounds offormula I, and compositions of the invention, respectively, i.e.enhanced health of the plant, are also present when the plant is notunder biotic stress and in particular when the plant is not under pestpressure.

For example, for seed treatment and soil applications, it is evidentthat a plant suffering from fungal or insecticidal attack shows reducedgermination and emergence leading to poorer plant or crop establishmentand vigor, and consequently, to a reduced yield as compared to a plantpropagation material which has been subjected to curative or preventivetreatment against the relevant pest and which can grow without thedamage caused by the biotic stress factor. However, the methodsaccording to the invention lead to an enhanced plant health even in theabsence of any biotic stress. This means that the positive effects ofthe strains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively,cannot be explained just by the pesticidal activities of strains, wholeculture broths, cell-free extracts, culture media, compounds of formulaI, and compositions of the invention, respectively, but are based onfurther activity profiles. Accordingly, the application of the strains,whole culture broths, cell-free extracts, culture media, compounds offormula I, and compositions of the invention, respectively, can also becarried out in the absence of pest pressure.

In an equally preferred embodiment, the present invention relates to amethod for improving the health of plants grown from said plantpropagation material, wherein the plant propagation material is treatedwith an effective amount of at least one strains, whole culture broths,cell-free extract, culture medium, compound of formula I, or acomposition of the invention.

Each plant health indicator listed below, which is selected from thegroups consisting of yield, plant vigor, quality and tolerance of theplant to abiotic and/or biotic stress, is to be understood as apreferred embodiment of the present invention either each on its own orpreferably in combination with each other.

According to the present invention, “increased yield” of a plant meansthat the yield of a product of the respective plant is increased by ameasurable amount over the yield of the same product of the plantproduced under the same conditions, but without the application of thestrains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively.

For seed treatment e. g. as inoculant and/or foliar application forms,increased yield can be characterized, among others, by the followingimproved properties of the plant: increased plant weight; and/orincreased plant height; and/or increased biomass such as higher overallfresh weight (FW); and/or increased number of flowers per plant; and/orhigher grain and/or fruit yield; and/or more tillers or side shoots(branches); and/or larger leaves; and/or increased shoot growth; and/orincreased protein content; and/or increased oil content; and/orincreased starch content; and/or increased pigment content; and/orincreased chlorophyll content (chlorophyll content has a positivecorrelation with the plant's photosynthesis rate and accordingly, thehigher the chlorophyll content the higher the yield of a plant) and/orincreased quality of a plant.

“Grain” and “fruit” are to be understood as any plant product which isfurther utilized after harvesting, e. g. fruits in the proper sense,vegetables, nuts, grains, seeds, wood (e. g. in the case of silvicultureplants), flowers (e. g. in the case of gardening plants, ornamentals)etc., that is anything of economic value that is produced by the plant.

According to the present invention, the yield is increased by at least4%. In general, the yield increase may even be higher, for example 5 to10%, more preferable by 10 to 20%, or even 20 to 30% According to thepresent invention, the yield—if measured in the absence of pestpressure—is increased by at least 2% In general, the yield increase mayeven be higher, for example until 4% to 5% or even more.

Another indicator for the condition of the plant is the plant vigor. Theplant vigor becomes manifest in several aspects such as the generalvisual appearance.

For foliar applications, improved plant vigor can be characterized,among others, by the following improved properties of the plant:improved vitality of the plant; and/or improved plant growth; and/orimproved plant development; and/or improved visual appearance; and/orimproved plant stand (less plant verse/lodging and/or bigger leaf blade;and/or bigger size; and/or increased plant height; and/or increasedtiller number; and/or increased number of side shoots; and/or increasednumber of flowers per plant; and/or increased shoot growth; and/orenhanced photosynthetic activity (e. g. based on increased stomatalconductance and/or increased CO₂ assimilation rate)); and/or earlierflowering; and/or earlier fruiting; and/or earlier grain maturity;and/or less non-productive tillers; and/or less dead basal leaves;and/or less input needed (such as fertilizers or water); and/or greenerleaves; and/or complete maturation under shortened vegetation periods;and/or easier harvesting; and/or faster and more uniform ripening;and/or longer shelf-life; and/or longer panicles; and/or delay ofsenescence; and/or stronger and/or more productive tillers; and/orbetter extractability of ingredients; and/or improved quality of seeds(for being seeded in the following seasons for seed production); and/orreduced production of ethylene and/or the inhibition of its reception bythe plant.

Another indicator for the condition of the plant is the “quality” of aplant and/or its products. According to the present invention, enhancedquality means that certain plant characteristics such as the content orcomposition of certain ingredients are increased or improved by ameasurable or noticeable amount over the same factor of the plantproduced under the same conditions, but without the application of thestrains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively.Enhanced quality can be characterized, among others, by followingimproved properties of the plant or its product: increased nutrientcontent; and/or increased protein content; and/or increased oil content;and/or increased starch content; and/or increased content of fattyacids; and/or increased metabolite content; and/or increased carotenoidcontent; and/or increased sugar content; and/or increased amount ofessential amino acids; and/or improved nutrient composition; and/orimproved protein composition; and/or improved composition of fattyacids; and/or improved metabolite composition; and/or improvedcarotenoid composition; and/or improved sugar composition; and/orimproved amino acids composition; and/or improved or optimal fruitcolor; and/or improved leaf color; and/or higher storage capacity;and/or better processability of the harvested products.

Another indicator for the condition of the plant is the plant'stolerance or resistance to biotic and/or abiotic stress factors. Bioticand abiotic stress, especially over longer terms, can have harmfuleffects on plants.

Biotic stress is caused by living organisms while abiotic stress iscaused for example by environmental extremes. According to the presentinvention, “enhanced tolerance or resistance to biotic and/or abioticstress factors” means (1.) that certain negative factors caused bybiotic and/or abiotic stress are diminished in a measurable ornoticeable amount as compared to plants exposed to the same conditions,but without being treated with the strains, whole culture broths,cell-free extracts, culture media, compounds of formula I, andcompositions of the invention, respectively, and (2.) that the negativeeffects are not diminished by a direct action of the strains, wholeculture broths, cell-free extracts, culture media, compounds of formulaI, and compositions of the invention, respectively, on the stressfactors, e. g. by its fungicidal or insecticidal action which directlydestroys the microorganisms or pests, but rather by a stimulation of theplants' own defensive reactions against said stress factors.

Negative factors caused by biotic stress such as pathogens and pests arewidely known and are caused by living organisms, such as competingplants (for example weeds), microorganisms (such as phytopathogenicfungi and/or bacteria) and/or viruses.

Negative factors caused by abiotic stress are also well-known and canoften be observed as reduced plant vigor (see above), for example:

less yield and/or less vigor, for both effects examples can be burnedleaves, less flowers, premature ripening, later crop maturity, reducednutritional value amongst others.

Abiotic stress can be caused for example by: extremes in temperaturesuch as heat or cold (heat stress/cold stress); and/or strong variationsin temperature; and/or temperatures unusual for the specific season;and/or drought (drought stress); and/or extreme wetness; and/or highsalinity (salt stress); and/or radiation (for example by increased UVradiation due to the decreasing ozone layer); and/or increased ozonelevels (ozone stress); and/or organic pollution (for example byphytotoxic amounts of pesticides); and/or inorganic pollution (forexample by heavy metal contaminants).

As a result of biotic and/or abiotic stress factors, the quantity andthe quality of the stressed plants decrease. As far as quality (asdefined above) is concerned, reproductive development is usuallyseverely affected with consequences on the crops which are important forfruits or seeds. Synthesis, accumulation and storage of proteins aremostly affected by temperature; growth is slowed by almost all types ofstress; polysaccharide synthesis, both structural and storage is reducedor modified: these effects result in a decrease in biomass (yield) andin changes in the nutritional value of the product.

As pointed out above, the above identified indicators for the healthcondition of a plant may be interdependent and may result from eachother. For example, an increased resistance to biotic and/or abioticstress may lead to a better plant vigor, e. g. to better and biggercrops, and thus to an increased yield. Inversely, a more developed rootsystem may result in an increased resistance to biotic and/or abioticstress. However, these interdependencies and interactions are neitherall known nor fully understood and therefore the different indicatorsare described separately.

In one embodiment the strains, whole culture broths, cell-free extracts,culture media, compounds of formula I, and compositions of theinvention, respectively, effectuate an increased yield of a plant or itsproduct. In another embodiment the strains, whole culture broths,cell-free extracts, culture media, compounds of formula I, andcompositions of the invention, respectively, effectuate an increasedvigor of a plant or its product. In another embodiment the strains,whole culture broths, cell-free extracts, culture media, compounds offormula I, and compositions of the invention, respectively, effectuatein an increased quality of a plant or its product. In yet anotherembodiment the strains, whole culture broths, cell-free extracts,culture media, compounds of formula I, and compositions of theinvention, respectively, effectuate an increased tolerance and/orresistance of a plant or its product against biotic stress. In yetanother embodiment the strains, whole culture broths, cell-freeextracts, culture media, compounds of formula I, and compositions of theinvention, respectively, effectuate an increased tolerance and/orresistance of a plant or its product against abiotic stress.

The strains, whole culture broths, cell-free extracts, culture media andcompounds of formula I, respectively, are employed as such or in form ofcompositions by treating the fungi or the plants, plant propagationmaterials, such as seeds, soil, surfaces, materials or rooms to beprotected from fungal attack with a fungicidally effective amount of theactive substances. The application can be carried out both before andafter the infection of the plants, plant propagation materials, such asseeds, soil, surfaces, materials or rooms by the fungi.

The term “effective amount” denotes an amount which is sufficient forcontrolling harmful fungi on cultivated plants or in the protection ofmaterials and which does not result in a substantial damage to thetreated plants. Such an amount can vary in a broad range and isdependent on various factors, such as the fungal species to becontrolled, the treated cultivated plant or material, the climaticconditions and the strains, whole culture broths, cell-free extracts,culture media and compounds of formula I or salt thereof, of theinvention, respectively, used.

Plant propagation materials may be treated with the strains, wholeculture broths, cell-free extracts, culture media, compounds of formulaI, and compositions of the invention, respectively, prophylacticallyeither at or before planting or transplanting.

The strains of the invention can be formulated as an inoculant for aplant. The term “inoculant” means a composition that includes anisolated strain of the invention and optionally a carrier, which mayinclude a biologically acceptable medium.

Such inoculants and other suitable compositions can be prepared ascompositions comprising besides the active ingredients at least oneauxiliary (inert ingredient) by usual means (see e. g. H. D. Burges:Formulation of Microbial Biopesticides, Springer, 1998).

To produce a dry formulation, bacterial cells, preferably spores can besuspended in a suitable dry carrier (e. g. clay). To produce a liquidformulation, cells, preferably spores, can be re-suspended in a suitableliquid carrier (e. g. water-based) to the desired spore density. Thespore density number of spores per ml can be determined by identifyingthe number of colony-forming units (CFU) on agar medium e. g. potatodextrose agar after incubation for several days at temperatures of about20 to about 30° C.

According to one embodiment, individual components of the compositionaccording to the invention such as parts of a kit or parts of a binaryor ternary mixture may be mixed by the user himself in a spray tank orany other kind of vessel used for applications (e.g seed treater drums,seed pelleting machinery, knapsack sprayer) and further auxiliaries maybe added, if appropriate. When living microorganisms, such as thePaenibacillus strains of the invention, form part of such kit, it mustbe taken care that choice and amounts of the other parts of the kit (e.g. chemical pesticidal agents) and of the further auxiliaries should notinfluence the viability of the microbial pesticides in the compositionmixed by the user. Especially for bactericides and solvents,compatibility with the respective microbial pesticide has to be takeninto account.

The strains, whole culture broths, cell-free extracts, culture mediaand/or compounds of formula I of the invention can be converted intocustomary types of agrochemical compositions, e. g. solutions,emulsions, suspensions, dusts, powders, pastes, granules, pressings,capsules, and mixtures thereof. Examples for composition types aresuspensions (e. g. SC, OD, FS), emulsifiable concentrates (e. g. EC),emulsions (e. g. EW, EO, ES, ME), capsules (e. g. CS, ZC), pastes,pastilles, wettable powders or dusts (e. g. WP, SP, WS, DP, DS),pressings (e. g. BR, TB, DT), granules (e. g. WG, SG, GR, FG, GG, MG),insecticidal articles (e. g. LN), as well as gel formulations for thetreatment of plant propagation materials such as seeds (e. g. GF). Theseand further compositions types are defined in the “Catalogue ofpesticide formulation types and international coding system”, TechnicalMonograph No. 2, 6^(th) Ed. May 2008, CropLife International.

The compositions are prepared in a known manner, such as described byMollet and Grubemann, Formulation technology, Wiley VCH, Weinheim, 2001;or Knowles, New developments in crop protection product formulation,Agrow Reports DS243, T&F Informa, London, 2005.

Suitable auxiliaries are solvents, liquid carriers, solid carriers orfillers, surfactants, dispersants, emulsifiers, wetters, adjuvants,solubilizers, penetration enhancers, protective colloids, adhesionagents, thickeners, humectants, repellents, attractants, feedingstimulants, compatibilizers, bactericides, anti-freezing agents,anti-foaming agents, colorants, tackifiers and binders.

Suitable solvents and liquid carriers are water and organic solvents,such as mineral oil fractions of medium to high boiling point, e. g.kerosene, diesel oil; oils of vegetable or animal origin;

aliphatic, cyclic and aromatic hydrocarbons, e. g. toluene, paraffin,tetrahydronaphthalene, alkylated naphthalenes; alcohols, e. g. ethanol,propanol, butanol, benzylalcohol, cyclohexanol; glycols; DMSO; ketones,e. g. cyclohexanone; esters, e. g. lactates, carbonates, fatty acidesters, gamma-butyrolactone; fatty acids; phosphonates; amines; amides,e. g. N-methylpyrrolidone, fatty acid dimethylamides; and mixturesthereof.

Suitable solid carriers or fillers are mineral earths, e. g. silicates,silica gels, talc, kaolins, limestone, lime, chalk, clays, dolomite,diatomaceous earth, bentonite, calcium sulfate, magnesium sulfate,magnesium oxide; polysaccharides, e. g. cellulose, starch; fertilizers,e. g. ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas;products of vegetable origin, e. g. cereal meal, tree bark meal, woodmeal, nutshell meal, and mixtures thereof.

Suitable surfactants are surface-active compounds, such as anionic,cationic, nonionic and amphoteric surfactants, block polymers,polyelectrolytes, and mixtures thereof. Such surfactants can be used asemusifier, dispersant, solubilizer, wetter, penetration enhancer,protective colloid, or adjuvant. Examples of surfactants are listed inMcCutcheon's, Voll: Emulsifiers & Detergents, McCutcheon's Directories,Glen Rock, USA, 2008 (International Ed. or North American Ed.).

Suitable anionic surfactants are alkali, alkaline earth or ammoniumsalts of sulfonates, sulfates, phosphates, carboxylates, and mixturesthereof. Examples of sulfonates are alkylarylsulfonates,diphenylsulfonates, alpha-olefin sulfonates, lignine sulfonates,sulfonates of fatty acids and oils, sulfonates of ethoxylatedalkylphenols, sulfonates of alkoxylated arylphenols, sulfonates ofcondensed naphthalenes, sulfonates of dodecyl- and tridecylbenzenes,sulfonates of naphthalenes and alkylnaphthalenes, sulfosuccinates orsulfosuccinamates. Examples of sulfates are sulfates of fatty acids andoils, of ethoxylated alkylphenols, of alcohols, of ethoxylated alcohols,or of fatty acid esters. Examples of phosphates are phosphate esters.Examples of carboxylates are alkyl carboxylates, and carboxylatedalcohol or alkylphenol ethoxylates.

Suitable nonionic surfactants are alkoxylates, N-substituted fatty acidamides, amine oxides, esters, sugar-based surfactants, polymericsurfactants, and mixtures thereof. Examples of alkoxylates are compoundssuch as alcohols, alkylphenols, amines, amides, arylphenols, fatty acidsor fatty acid esters which have been alkoxylated with 1 to 50equivalents. Ethylene oxide and/or propylene oxide may be employed forthe alkoxylation, preferably ethylene oxide. Examples of N-substitutedfatty acid amides are fatty acid glucamides or fatty acid alkanolamides.Examples of esters are fatty acid esters, glycerol esters ormonoglycerides. Examples of sugar-based surfactants are sorbitans,ethoxylated sorbitans, sucrose and glucose esters oralkylpolyglucosides. Examples of polymeric surfactants are home- orcopolymers of vinylpyrrolidone, vinyl alcohols, or vinyl acetate.

Suitable cationic surfactants are quaternary surfactants, for examplequaternary ammonium compounds with one or two hydrophobic groups, orsalts of long-chain primary amines. Suitable amphoteric surfactants arealkylbetains and imidazolines. Suitable block polymers are blockpolymers of the A-B or A-B-A type comprising blocks of polyethyleneoxide and polypropylene oxide, or of the A-B-C type comprising alkanol,polyethylene oxide and polypropylene oxide. Suitable polyelectrolytesare polyacids or polybases. Examples of polyacids are alkali salts ofpolyacrylic acid or polyacid comb polymers. Examples of polybases arepolyvinyl amines or polyethyleneamines.

Suitable adjuvants are compounds, which have a negligible or even nopesticidal activity themselves, and which improve the biologicalperformance of cell-free extract, culture medium or metabolite on thetarget. Examples are surfactants, mineral or vegetable oils, and otherauxilaries. Further examples are listed by Knowles, Adjuvants andadditives, Agrow Reports DS256, T&F Informa UK, 2006, chapter 5.

Suitable thickeners are polysaccharides (e. g. xanthan gum,carboxymethyl cellulose), inorganic clays (organically modified orunmodified), polycarboxylates, and silicates.

Suitable bactericides are bronopol and isothiazolinone derivatives suchas alkylisothiazolinones and benzisothiazolinones. Suitableanti-freezing agents are ethylene glycol, propylene glycol, urea andglycerin. Suitable anti-foaming agents are silicones, long chainalcohols, and salts of fatty acids. Suitable colorants (e. g. in red,blue, or green) are pigments of low water solubility and water-solubledyes. Examples are inorganic colorants (e. g. iron oxide, titan oxide,iron hexacyanoferrate) and organic colorants (e. g. alizarin-, azo- andphthalocyanine colorants). Suitable tackifiers or binders are polyvinylpyrrolidones, polyvinyl acetates, polyvinyl alcohols, polyacrylates,biological or synthetic waxes, and cellulose ethers.

When living microorganisms, such as Paenibacillus strains of theinvention in form of cells or spores, form part of the compositions,such compositions can be prepared as compositions comprising besides theactive ingredients at least one auxiliary (inert ingredient) by usualmeans (see e. g. H. D. Burges: Formulation of Microbial Biopesticides,Springer, 1998). Suitable customary types of such compositions aresuspensions, dusts, powders, pastes, granules, pressings, capsules, andmixtures thereof. Examples for composition types are suspensions (e. g.SC, OD, FS), capsules (e. g. CS, ZC), pastes, pastilles, wettablepowders or dusts (e. g. WP, SP, WS, DP, DS), pressings (e. g. BR, TB,DT), granules (e. g. WG, SG, GR, FG, GG, MG), insecticidal articles (e.g. LN), as well as gel formulations for the treatment of plantpropagation materials such as seeds (e. g. GF). Herein, it has to betaken into account that each formulation type or choice of auxiliaryshould not influence the viability of the microorganism during storageof the composition and when finally applied to the soil, plant or plantpropagation material. Suitable formulations are e. g. mentioned in WO2008/002371, U.S. Pat. Nos. 6,955,912, 5,422,107.

Examples for suitable auxiliaries are those mentioned earlier herein,wherein it must be taken care that choice and amounts of suchauxiliaries should not influence the viability of the microbialpesticides in the composition. Especially for bactericides and solvents,compatibility with the respective microorganism of the respectivemicrobial pesticide has to be taken into account. In addition,compositions with microbial pesticides may further contain stabilizersor nutrients and UV protectants. Suitable stabilizers or nutrients aree. g. alpha-tocopherol, trehalose, glutamate, potassium sorbate, varioussugars like glucose, sucrose, lactose and maltodextrine Burges:Formulation of Microbial Biopesticides, Springer, 1998). Suitable UVprotectants are e. g. inorganic compounds like titanium dioxide, zincoxide and iron oxide pigments or organic compounds like benzophenones,benzotriazoles and phenyltriazines. The compositions may in addition toauxiliaries mentioned for compositions comprising compounds I hereinoptionally comprise 0.1-80% stabilizers or nutrients and 0.1-10% UVprotectants.

The agrochemical compositions generally comprise between 0.01 and 95%,preferably between 0.1 and 90%, and in particular between 0.5 and 75%,by weight of active substance. The active substances are employed in apurity of from 90% to 100%, preferably from 95% to 100% (according toNMR spectrum).

Examples for composition types and their preparation are:

i) Water-Soluble Concentrates (SL, LS)

10-60 wt % of a whole culture broth, cell-free extract, culture mediumor metabolite of the invention and 5-15 wt % wetting agent (e. g.alcohol alkoxylates) are dissolved in water and/or in a water-solublesolvent (e. g. alcohols) ad 100 wt %. The active substance dissolvesupon dilution with water.

ii) Dispersible Concentrates (DC)

5-25 wt % of a whole culture broth, cell-free extract, culture medium ormetabolite of the invention and 1-10 wt % dispersant (e. g. polyvinylpyrrolidone) are dissolved in organic solvent (e. g. cyclohexanone) ad100 wt %. Dilution with water gives a dispersion.

iii) Emulsifiable Concentrates (EC)

15-70 wt % of a whole culture broth, cell-free extract, culture mediumor metabolite of the invention and 5-10 wt % emulsifiers (e. g. calciumdodecylbenzenesulfonate and castor oil ethoxylate) are dissolved inwater-insoluble organic solvent (e. g. aromatic hydrocarbon) ad 100 wt%. Dilution with water gives an emulsion.

iv) Emulsions (EW, EO, ES)

5-40 wt % of a whole culture broth, cell-free extract, culture medium ormetabolite of the invention and 1-10 wt % emulsifiers (e. g. calciumdodecylbenzenesulfonate and castor oil ethoxylate) are dissolved in20-40 wt % water-insoluble organic solvent (e. g. aromatic hydrocarbon).This mixture is introduced into water ad 100 wt % by means of anemulsifying machine and made into a homogeneous emulsion. Dilution withwater gives an emulsion.

v) Suspensions (SC, OD, FS)

In an agitated ball mill, 20-60 wt % of a whole culture broth, cell-freeextract, culture medium or metabolite of the invention are comminutedwith addition of 2-10 wt % dispersants and wetting agents (e. g. sodiumlignosulfonate and alcohol ethoxylate), 0.1-2 wt % thickener (e. g.xanthan gum) and water ad 100 wt % to give a fine active substancesuspension. Dilution with water gives a stable suspension of the activesubstance. For FS type composition up to 40 wt % binder (e. g. polyvinylalcohol) is added.

vi) Water-Dispersible Granules and Water-Soluble Granules (WG, SG)

50-80 wt % of a whole culture broth, cell-free extract, culture mediumor metabolite of the invention are ground finely with addition ofdispersants and wetting agents (e. g. sodium lignosulfonate and alcoholethoxylate) ad 100 wt % and prepared as water-dispersible orwater-soluble granules by means of technical appliances (e. g.extrusion, spray tower, fluidized bed). Dilution with water gives astable dispersion or solution of the active substance.

vii) Water-Dispersible Powders and Water-Soluble Powders (WP, SP, WS)

50-80 wt % of a whole culture broth, cell-free extract, culture mediumor metabolite of the invention are ground in a rotor-stator mill withaddition of 1-5 wt % dispersants (e. g. sodium lignosulfonate), 1-3 wt %wetting agents (e. g. alcohol ethoxylate) and solid carrier (e. g.silica gel) ad 100 wt %. Dilution with water gives a stable dispersionor solution of the active substance.

viii) Gel (GW, GF)

In an agitated ball mill, 5-25 wt % of a whole culture broth, cell-freeextract, culture medium or metabolite of the invention are comminutedwith addition of 3-10 wt % dispersants (e. g. sodium lignosulfonate),1-5 wt % thickener (e. g. carboxymethyl cellulose) and water ad 100 wt %to give a fine suspension of the active substance. Dilution with watergives a stable suspension of the active substance.

ix) Microemulsion (ME)

5-20 wt % of a whole culture broth, cell-free extract, culture medium ormetabolite of the invention are added to 5-30 wt % organic solvent blend(e. g. fatty acid dimethylamide and cyclohexanone), 10-25 wt %surfactant blend (e. g. alcohol ethoxylate and arylphenol ethoxylate),and water ad 100%. This mixture is stirred for 1 h to producespontaneously a thermodynamically stable microemulsion.

x) Microcapsules (CS)

An oil phase comprising 5-50 wt % of a whole culture broth, cell-freeextract, culture medium or metabolite of the invention, 0-40 wt % waterinsoluble organic solvent (e. g. aromatic hydrocarbon), 2-15 wt %acrylic monomers (e. g. methylmethacrylate, methacrylic acid and a di-or triacrylate) are dispersed into an aqueous solution of a protectivecolloid (e. g. polyvinyl alcohol). Radical polymerization initiated by aradical initiator results in the formation of poly(meth)acrylatemicrocapsules.

Alternatively, an oil phase comprising 5-50 wt % of a whole culturebroth, cell-free extract, culture medium or metabolite of the invention,0-40 wt % water insoluble organic solvent (e. g. aromatic hydrocarbon),and an isocyanate monomer (e. g. diphenylmethene-4,4′-diisocyanatae) aredispersed into an aqueous solution of a protective colloid (e. g.polyvinyl alcohol). The addition of a polyamine (e. g.hexamethylenediamine) results in the formation of polyureamicrocapsules. The monomers amount to 1-10 wt %. The wt % relate to thetotal CS composition.

xi) Dustable Powders (DP, DS)

1-10 wt % of a whole culture broth, cell-free extract, culture medium ormetabolite of the invention are ground finely and mixed intimately withsolid carrier (e. g. finely divided kaolin) ad 100 wt %.

xii) Granules (GR, FG)

0.5-30 wt % of a whole culture broth, cell-free extract, culture mediumor metabolite of the invention are ground finely and associated withsolid carrier (e. g. silicate) ad 100 wt %. Granulation is achieved byextrusion, spray-drying or fluidized bed.

xiii) Ultra-Low Volume Liquids (UL)

1-50 wt % of a whole culture broth, cell-free extract, culture medium ormetabolite of the invention are dissolved in organic solvent (e. g.aromatic hydrocarbon) ad 100 wt %.

The compositions types i) to xiii) may optionally comprise furtherauxiliaries, such as 0.1-1 wt % bactericides, 5-15 wt % anti-freezingagents, 0.1-1 wt % anti-foaming agents, and 0.1-1 wt % colorants.

Solutions for seed treatment (LS), suspoemulsions (SE), flowableconcentrates (FS), powders for dry treatment (DS), water-dispersiblepowders for slurry treatment (WS), water-soluble powders (SS), emulsions(ES), emulsifiable concentrates (EC) and gels (GF) are usually employedfor the purposes of treatment of plant propagation materials,particularly seeds.

Preferred examples of seed treatment formulation types or soilapplication for pre-mix compositions are of WS, LS, ES, FS, WG orCS-type.

Typically, a pre-mix formulation for seed treatment applicationcomprises 0.5 to 99.9 percent, especially 1 to 95 percent, of thedesired ingredients, and 99.5 to 0.1 percent, especially 99 to 5percent, of a solid or liquid adjuvant (including, for example, asolvent such as water), where the auxiliaries can be a surfactant in anamount of 0 to 50 percent, especially 0.5 to 40 percent, based on thepre-mix formulation. Whereas commercial products will preferably beformulated as concentrates (e. g., pre-mix composition (formulation)),the end user will normally employ dilute formulations (e. g., tank mixcomposition).

Seed treatment methods for applying or treating the strains, wholeculture broths, cell-free extracts, culture media, compounds of formulaI and compositions of the invention, respectively, to plant propagationmaterial, especially seeds, are known in the art, and include dressing,coating, filmcoating, pelleting and soaking application methods of thepropagation material. Such methods are also applicable to thecombinations according to the invention. In a preferred embodiment, thestrains, whole culture broths, cell-free extracts, culture media,compounds of formula I, and compositions of the invention, respectively,are applied or treated onto the plant propagation material by a methodsuch that the germination is not negatively impacted. Accordingly,examples of suitable methods for applying (or treating) a plantpropagation material, such as a seed, is seed dressing, seed coating orseed pelleting and alike.

It is preferred that the plant propagation material is a seed, seedpiece (i.e. stalk) or seed bulb.

Although it is believed that the present method can be applied to a seedin any physiological state, it is preferred that the seed be in asufficiently durable state that it incurs no damage during the treatmentprocess. Typically, the seed would be a seed that had been harvestedfrom the field; removed from the plant; and separated from any cob,stalk, outer husk, and surrounding pulp or other non-seed plantmaterial. The seed would preferably also be biologically stable to theextent that the treatment would cause no biological damage to the seed.It is believed that the treatment can be applied to the seed at any timebetween harvest of the seed and sowing of the seed or during the sowingprocess (seed directed applications). The seed may also be primed eitherbefore or after the treatment.

Even distribution of the ingredients in the strains, whole culturebroths, cell-free extracts, culture media, compounds of formula I, andcompositions of the invention, respectively, and adherence thereof tothe seeds is desired during propagation material treatment. Treatmentcould vary from a thin film (dressing) of the formulation containing thecombination, for example, a mixture of active ingredient(s), on a plantpropagation material, such as a seed, where the original size and/orshape are recognizable to an intermediary state (such as a coating) andthen to a thicker film (such as pelleting with many layers of differentmaterials (such as carriers, for example, clays; different formulations,such as of other active ingredients; polymers; and colourants) where theoriginal shape and/or size of the seed is no longer recognizable.

An aspect of the present invention includes application of the strains,whole culture broths, cell-free extracts, culture media, compounds offormula I, and compositions of the invention, respectively, onto theplant propagation material in a targeted fashion, including positioningthe ingredients in the combination onto the entire plant propagationmaterial or on only parts thereof, including on only a single side or aportion of a single side. One of ordinary skill in the art wouldunderstand these application methods from the description provided inEP954213B1 and WO06/112700.

The strains, whole culture broths, cell-free extracts, culture media,compounds of formula I and compositions of the invention, respectively,can also be used in form of a “pill” or “pellet” or a suitable substrateand placing, or sowing, the treated pill, or substrate, next to a plantpropagation material. Such techniques are known in the art, particularlyin EP1124414, WO07/67042, and WO 07/67044. Application of the strains,whole culture broths, cell-free extracts, culture media, compounds offormula I and compositions, respectively, described herein onto plantpropagation material also includes protecting the plant propagationmaterial treated with the combination of the present invention byplacing one or more pesticide-containing particles next to apesticide-treated seed, wherein the amount of pesticide is such that thepesticide-treated seed and the pesticide-containing particles togethercontain an Effective Dose of the pesticide and the pesticide dosecontained in the pesticide-treated seed is less than or equal to theMaximal Non-Phytotoxic Dose of the pesticide. Such techniques are knownin the art, particularly in WO2005/120226.

Application of the strains, whole culture broths, cell-free extracts,culture media, compounds of formula I and compositions of the invention,respectively, onto the seed also includes controlled release coatings onthe seeds, wherein the ingredients of the combinations are incorporatedinto materials that release the ingredients over time. Examples ofcontrolled release seed treatment technologies are generally known inthe art and include polymer films, waxes, or other seed coatings,wherein the ingredients may be incorporated into the controlled releasematerial or applied between layers of materials, or both.

Seed can be treated by applying thereto the strains, whole culturebroths, cell-free extracts, culture media, compounds of formula I, andcompositions of the invention, respectively, in any desired sequence orsimultaneously.

The seed treatment occurs to an unsown seed, and the term “unsown seed”is meant to include seed at any period between the harvest of the seedand the sowing of the seed in the ground for the purpose of germinationand growth of the plant.

Treatment to an unsown seed is not meant to include those practices inwhich the active ingredient is applied to the soil but would include anyapplication practice that would target the seed during the plantingprocess.

Preferably, the treatment occurs before sowing of the seed so that thesown seed has been pre-treated with the strains, whole culture broths,cell-free extracts, culture media, compounds of formula I andcompositions of the invention, respectively. In particular, seed coatingor seed pelleting are preferred. As a result of the treatment, theingredients are adhered on to the seed and therefore available for pestcontrol.

The treated seeds can be stored, handled, sowed and tilled in the samemanner as any other active ingredient treated seed.

In particular, the present invention relates to a method for protectionof plant propagation material from pests and/or improving the health ofplants grown from said plant propagation material, wherein the soil,wherein plant propagation material is sown, is treated with an effectiveamount of a strain, cell-free extract, culture medium, metabolite orcomposition of the invention, respectively.

In particular, the present invention relates to a method for protectionof plant propagation material from pests, wherein the soil, whereinplant propagation material is sown, is treated with an effective amountof a strain, cell-free extract, culture medium, metabolite orcomposition of the invention, respectively.

In particular, the present invention relates to a method for protectionof plant propagation material from harmful fungi, wherein the soil,wherein plant propagation material is sown, is treated with an effectiveamount of a strain, cell-free extract, culture medium, metabolite orcomposition of the invention, respectively.

In particular, the present invention relates to a method for protectionof plant propagation material from animal pests (insects, acarids ornematodes), wherein the soil, wherein plant propagation material issown, is treated with an effective amount of a strain, cell-freeextract, culture medium, metabolite or composition of the invention,respectively.

The user applies the compositions of the invention usually from apredosage device, a knapsack sprayer, a spray tank, a spray plane, or anirrigation system. Usually, the agrochemical composition is made up withwater, buffer, and/or further auxiliaries to the desired applicationconcentration and the ready-to-use spray liquor or the agrochemicalcomposition according to the invention is thus obtained. Usually, 20 to2000 liters, preferably 50 to 400 liters, of the ready-to-use sprayliquor are applied per hectare of agricultural useful area.

When it comes to the treatment of plant propagation material, especiallyseeds, the compositions disclosed herein give, after two-to-tenfolddilution, active components concentrations of from 0.01 to 60% byweight, preferably from 0.1 to 40%, in the ready-to-use preparations.Application can be carried out before or during sowing. Methods forapplying a strain, cell-free extract, culture medium, metabolite orcomposition of the invention, respectively, onto plant propagationmaterial, especially seeds, include dressing, coating, pelleting,dusting, soaking and in-furrow application methods of the propagationmaterial. Preferably, the strains, whole culture broths, cell-freeextracts, culture media, compounds of formula I or compositions of theinvention, respectively, are applied onto the plant propagation materialby a method such that germination is not induced, e. g. by seeddressing, pelleting, coating and dusting.

When the strains of the invention are employed in crop protection,wherein the strains are applied as foliar treatment or to the soil, theapplication rates usually range from about 1×10⁶ to 5×10¹⁵ (or more)CFU/ha, preferably from about 1×10⁷ to about 1×10¹³ CFU/ha, even morepreferably from 1×10⁹ to 5×10¹² CFU/ha.

When the strains of the invention are employed in seed treatment, theapplication rates with respect to plant propagation material usuallyrange from about 1×10¹ to 1×10¹² (or more) CFU/seed, preferably fromabout 1×10³ to about 1×10¹⁰ CFU/seed, and even more preferably fromabout 1×10³ to about 1×10⁶ CFU/seed. Alternatively, the applicationrates with respect to plant propagation material preferably range fromabout 1×10⁷ to 1×10¹⁶ (or more) CFU per 100 kg of seed, preferably from1×10⁹ to about 1×10¹⁵ CFU per 100 kg of seed, even more preferably from1×10¹¹ to about 1×10¹⁵ CFU per 100 kg of seed.

When cell-free extracts, culture media and/or compounds of formula I areemployed, the solid material (dry matter) are considered as activecomponents, e. g. to be obtained after drying or evaporation of theextraction medium or the suspension medium in case of liquidformulations. When employed in plant protection, the amounts of activecomponents applied are, depending on the kind of effect desired, from0.001 to 2 kg per ha, preferably from 0.005 to 2 kg per ha, morepreferably from 0.05 to 0.9 kg per ha, and in particular from 0.1 to0.75 kg per ha. In treatment of plant propagation materials such asseeds, e. g. by dusting, coating or drenching seed, amounts of activecomponents of from 0.1 to 1000 g, preferably from 1 to 1000 g, morepreferably from 1 to 100 g and most preferably from 5 to 100 g, per 100kilogram of plant propagation material (preferably seeds) are generallyrequired. When used in the protection of materials or stored products,the amount of active components applied depends on the kind ofapplication area and on the desired effect. Amounts customarily appliedin the protection of materials are 0.001 g to 2 kg, preferably 0.005 gto 1 kg, of active components per cubic meter of treated material.

According to one embodiment, individual components of the composition ofthe invention such as parts of a kit or parts of a binary or ternarymixture may be mixed by the user himself in a spray tank or any otherkind of vessel used for applications (e.g seed treater drums, seedpelleting machinery, knapsack sprayer) and further auxiliaries may beadded, if appropriate.

If living microorganisms, such as the strains of the invention, formpart of such kit, it must be taken care that choice and amounts of thecomponents (e. g. chemical pesticidal agents) and of the furtherauxiliaries should not influence the viability of the microorganisms inthe composition mixed by the user. Especially for bactericides andsolvents, compatibility with the respective microorganisms has to betaken into account.

Various types of oils, wetters, adjuvants, fertilizer, ormicronutrients, and further pesticides (e. g. herbicides, insecticides,fungicides, growth regulators, safeners, biopesticides) may be added tothe strains, cell-free extracts, culture media, metabolites, compoundsof formula I and composition of the invention, respectively as premixor, if appropriate not until immediately prior to use (tank mix). Theseagents can be admixed with the compositions according to the inventionin a weight ratio of 1:100 to 100:1, preferably 1:10 to 10:1.Preferably, a composition of the invention comprises a furtherbiopesticide. Even more preferably, a composition of the inventioncomprises besides an auxiliary and at least one compound of formula I, amicrobial pesticide.

A pesticide is generally a chemical or biological agent (such as avirus, bacterium, antimicrobial or disinfectant) that through its effectdeters, incapacitates, kills or otherwise discourages pests. Targetpests can include insects, plant pathogens, weeds, mollusks, birds,mammals, fish, nematodes (roundworms), and microbes that destroyproperty, cause nuisance, spread disease or are vectors for disease. Theterm pesticides includes also plant growth regulators that alter theexpected growth, flowering, or reproduction rate of plants; defoliantsthat cause leaves or other foliage to drop from a plant, usually tofacilitate harvest; desiccants that promote drying of living tissues,such as unwanted plant tops; plant activators that activate plantphysiology for defense of against certain pests; safeners that reduceunwanted herbicidal action of pesticides on crop plants; and plantgrowth promoters that affect plant physiology to increase plant growth,biomass, yield or any other quality parameter of the harvestable goodsof a crop plant.

EXAMPLES

The present invention will be described in greater detail by means ofthe following examples. The following examples are for illustrativepurposes and are not intended to limit the scope of the invention.

Example 1: Isolation of Novel Bacterial Strains of the Invention

Soil samples from a variety of European locations including Germany werecollected. By applying commonly known microbial isolation procedures tothese soils, the inventors obtained a variety of bacteria that werefurther subjected to conventional isolation techniques for providingpure isolates as described herein.

Standard microbial enrichment technique (C. A. Reddy, T. J. Beveridge,J. A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R. Snyder (eds.).Methods for General and Molecular Microbiology, Am. Soc. Microbiol.,Washington, D.C.) was followed to isolate each type of bacteria.

The following strains have been isolated and deposited under BudapestTreaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) on Feb. 20, 2013:

a) Lu16774 as deposited with DSMZ having the deposit number DSM 26969

b) Lu17007 as deposited with DSMZ having the deposit number DSM 26970

c) Lu17015 as deposited with DSMZ having the deposit number DSM 26971.

Example 2—Characterization of Novel Bacterial Strains Example 2.1:16S-rDNA Sequencing

The 16S rRNA gene sequences of the Paenibacillus strains were determinedby direct sequencing of PCR-amplified 16S rDNA at the DSMZ,Braunschweig, Germany.

Genomic DNA extraction was carried out using the MasterPure™ GramPositive DNA Purification Kit from Epicentre Biotechnologies accordingto the manufacturer's instructions. PCR-mediated amplification of the16S rDNA and purification of the PCR product was carried out asdescribed previously (Int. J. Syst. Bacteriol. 46, 1088-1092, 1996).Purified PCR products were sequenced using the BigDye® Terminator v1.1Cycle Sequencing Kit (Applied Biosystems) as directed in themanufacturer's protocol. Sequence reactions were electrophoresed usingthe 3500xL Genetic Analyzer from Applied Biosystems. Sequenceambiguities may be due to the existence of several cistrons encoding 16SrRNAs with different sequences within a single genome (J. Bacteriol.178(19), 5636-5643, 1996).

The resulting sequence data from the strains was put into the alignmenteditor AE2 (http://iubio.bio.indiana.edu/soft/molbio/unix/ae2.readme),aligned manually according to the secondary structure of the resultingrRNA molecule and compared with representative 16S rRNA gene sequencesof organisms belonging to the Firmicutes (Nucl. Acids Res. 27, 171-173,1999). For comparison, 16S rRNA sequences were obtained from the EMBLand RDP data bases.

The 16S rDNA sequences of the strains of the invention are set forth inthe Sequence Listing as indicated in Table 2.

TABLE 2 Sequence listing references of the 16S rDNA of the Paenibacillusstrains. Strain SEQ ID NO Lu16774 1 Lu17007 2 Lu17015 3

The 16S rDNA gene identity values in % were calculated by pairwisecomparison of the sequences within the alignment of the sequencescompared.

Comparison performed of only two sequences based on pairwise sequencealignment are denoted herein as binary values. The other values arebased on a multiple sequence alignment of all sequences within thecomparison. Higher identity values from multi-sequence comparisonsresult from the problem that the sequence data of the compared sequenceswere of different length resulting in a shorter alignment.

The % identity from pair-wise comparisons of the complete rDNA sequencesamong the three novel strains Lu16774, Lu17007 and Lu17015 was between99.5 and 99.9% (Table 3, binary values).

TABLE 3 Identity in % of the complete 16S rRNA sequences of the threenovel Paenibacillus strains (binary values in brackets). Identity of thecomplete 16S rRNA sequence of the novel Paenibacillus strains (%)Strains Lu16774 Lu17015 Lu17007 Lu16774 — Lu17015 99.7 (99.5) — Lu1700799.9 (99.8) 99.8 (99.5) —

The comparison of the complete 16S rRNA sequence of the three novelstrains Lu16774, Lu17007 and Lu17015 with related taxa (see FIG. 9)revealed a high percentage of identity to Paenibacillus peoriae(type-strain DSM 8320) with 99.8%. The binary values forpairwise-sequence alignments of P. peoriae with the novel strains wereas follows: Lu16774: 99.5%, Lu17007: 99.5%; and Lu17015: 99.7% identity,respectively.

A final evaluation of species to which the novel Paenibacillus strainsLu16774, Lu17015 and Lu17007 belong was based on the 16S rRNA sequencedata not possible.

The sequencing of the complete rDNA resulted for Paenibacillus peoriaeNRRL BD-62 in 100.0% identity to P. peoriae (type strain DSM 8320)confirming the species designation P. peoriae for this strain BD-62 (seeFIG. 9).

The close relationship of all three novel Paenibacillus strains Lu16774,Lu17007 and Lu17015 to P. peoriae was confirmed by the comparison withthe 16S rRNA sequence of P. peoriae strain BD62 which resulted inidentity values of 99.8% (see FIG. 9).

For construction of the phylogenetic dendrogram operations of the ARBpackage (Nucl. Acids Res. 35, 7188-7196, 2007) were used: based on theevolutionary distance values the phylogenetic tree was constructed bythe neighbor-joining method (Jukes, T. H. & Cantor C. R. (1969).Evolution of protein molecules. In Mammalian protein metabolism, pp.21-132. Edited by H. N. Munro. New York: Academic press) using thecorrection of Jukes and Cantor (Mol. Biol. Evol. 4, 406-425, 1987). Theroot of the tree was determined by including the 16S rRNA gene sequenceof Cohnella thermotolerans into the analysis. The scale bar below thedendrogram indicates 1 nucleotide substitutions per 100 nucleotides. Theresults are given in FIG. 10.

The phylogenetic dendrogram of these sequences (FIG. 10) shows that thethree novel strains Lu16774, Lu17007 and Lu17015 are most-closelyrelated to each other and that their closest relative known to each ofthem was the Paenibacillus peoriae strain NRRL BD-62.

Example 2.2: RiboPrint-Analysis

Standardized, automated ribotyping is performed using the QualiconRiboPrintersystem. The RiboPrinter system combines molecular processingsteps for ribotyping in a stand-alone, automated instrument. Theprocedure includes cell lysis, digestion of chromosomal DNA withrestriction enzyme EcoRI, separation of fragments by electrophoresis,transfer of DNA fragments to a nylon membrane, hybridization to a probegenerated from the rrnB operon from E. coli, chemiluminescent detectionof the probe to the fragments containing rrn operon sequences, imagedetection and computerized analysis of RiboPrint patterns (FoodTechnology 50(1), 77-81, 1996; Proc. Natl. Acad. Sci. USA 92, 5229-5233,1995; Int. Journ. Syst. Bact. 44(3), 454-460, 1994).

Ribotyping have been executed by the DSMZ, Germany with the novelPaenibacillus strains Lu16774, Lu17007 and Lu17015 in comparison to theP. peoriae strain BD-62 using the restriction enzyme EcoRI. Theresulting patterns have been compared using the Software of theRiboPrinter system, the integrated DuPont Identification Library as wellas the BioNumerics Software (Applied Maths, Belgium).

Similarity of all three novel strains to BD-62 was between 0.24 and 0.5(FIG. 11). The three novel strains group in two groups, first comprisingLu17015, whereas the second group comprises the strains Lu16774 andLu17007. None of the novel strains has a similarity higher than 0.84 toany strain within the DuPont Identification Library and was thereforenot identified automatically.

The strain BD-62 has been identified as Paenibacillus peoriae based onthe entry DUP-13142 of the DuPont identification library (entry based onPaenibacillus peoriae DSM 8320).

Example 2.3: Morphological and Physiological Characterization

The strains were characterized at the DSMZ in analogy to methodsdescribed in Gordon, R. E., Haynes, W. C. & Pang. C. H.-N. (1973): TheGenus Bacillus, Agriculture Handbook no. 427. Washington D.C.: USDepartment of Agriculture. The results are given in Table 4.

TABLE 4 Characterization Data of the Paenibacillus strains of theinvention and comparison to known Paenibacillus peoriae strain NRRLBD-62. Paenibacillus strains Identification Lu16774 Lu17007 Lu17015BD-62 Characteristics cell form rod- rod- rod- rod- shaped shaped shapedshaped width [μm] 0.9-1.0 0.9-1.0 0.9-1.0 0.9-1.0 length [μm] 3−>5.0  3-5.0   3-5.0 2.5-5.0 ellipsoid spores + + + + swollensporangium + + + + Catalase + + + + Oxidase − − − − anaerobicgrowth + + + + VP reaction + + + + pH in VP-Medium 5.2 5.7 4.8 5.2maximum temperature positive growth at ° C. 40 40 40 40 negative growthat ° C. 50 50 50 50 Growth in: Medium pH 5.7 + + + + NaCl 2% + + + +NaCl 5% − − − − NaCl 7% − − − − Acid formation from: D-Glucose + + + +L-Arabinose + + + + D-Xylose + + + + D-Mannitol + + + +D-Fructose + + + + Raffinose + + + + Trehalose + + + − Glycerol + + + +Gas from glucose + + + + Hydrolysis of starch + + + + gelatin + + + +casein + + + ? Tween 80 − − − − esculin + + + + Utilisation of citraten.g.* n.g. n.g. n.g. propionate n.g.  n.g. n.g. n.g. N0₃ to N0₂ + + + +Indole reaction − − − − Lecithinase + + + − Phenylalanine − − − −desaminase Arginine dihydrolase − − − − Lysozyme + + + + *n.g. = nogrowth.

Analysis of the cellular fatty acids performed at the DSMZ resulted thatall strains showed at typical profile for Paenibacillus spp.

Using the available genetic, physiological and biochemical data, it isshown that the strains Lu16774, Lu17007 and Lu17015 belong to the genusPaenibacillus. As the strains Lu16774, Lu17007 and Lu17015 as well asBD-62 do produce gas from glucose, none of them belongs to Paenibacillusjamilae.

A phenotypic differentiation between Paenibacillus peoriae andPaenibacillus polymyxa is primarily possible using characteristics ofacid production from certain substrates (Int. J. Syst. Bacteriol. 43(2),388-390, 1993; In. J. Syst. Bacteriol. 46(6), 988-1003, 1996). None ofthe novel strains did completely match with its characteristics outlinedin Table 4 completely to any of these two species, but in sum of theavailable genetic, physiological and biochemical data most likely pointto the species Paenibacillus peoriae and P. polymyxa or at least toanother species very closely related to Paenibacillus peoriae and P.polymyxa.

Due to the multitude of Paenibacillus species described so far, it isimpossible to determine the correct taxonomic species of the threeisolates tested based on physiological and morphological criteria fromTable 4 (Rainer Borriss, Humboldt University Berlin, unpublishedresults).

Nevertheless, it was not possible to completely determine the specieswithin this genus. The most closely related species and strain was foundto be Paenibacillus peoriae BD-62 based on 16S-rDNA analysis (see e. g.FIG. 11).

Example 2.4: Phylogenetic Analysis Based on Genes Coding for DnaN, GyrB,RecF, RecN and RpoA

The nucleotide sequences of the genes coding for DnaN, GyrB, RecF, RecNand RpoA have been extracted from complete genome sequences or frompublic databases (Sequence listings as outlined in Table 28).

The identity tables (FIGS. 12 to 16) have been generated with an allagainst all approach where every sequence is aligned with every othersequence. The sequence alignment was performed with a program needle(EMBOSS package 6.6.0; Trends in Genetics 16 (6), 276-277). Standardparameters where used (gap creation 10.0; gap extension 0.5). IdentityScores are are calculated on the basis of the alignments without takingany gaps into account.

For the phylogenetic trees (FIGS. 17 to 21), multiple sequencealignments that have been performed with Clustal Omega (version 1.2.0;Molecular Systems Biology 7: 539, doi:10.1038/msb.2011.75). Thephylogenetic trees are calculated by maximum likelyhood method with thesoftware Dnaml (implemented in the Phylip 3.696 package; Felsenstein1981, http://evolution.genetics.washington.edu/phylip.html). Thedendrograms have been established using a F84 distance model whileapplying a transition-transversion ratio of two (2). The trees areplotted with the tool Dendroscope (http://dendroscope.org/).

TABLE 28 Sequence listing references of the dnaN, gyrB, recF, recN andrpoA DNA sequences of the Paenibacillus strains. Strain Gene SEQ ID NOLu16774 dnaN 4 Lu17007 dnaN 5 Lu17015 dnaN 6 Lu16774 gyrB 7 Lu17007 gyrB8 Lu17015 gyrB 9 Lu16774 recF 10 Lu17007 recF 11 Lu17015 recF 12 Lu16774recN 13 Lu17007 recN 14 Lu17015 recN 15 Lu16774 rpoA 16 Lu17007 rpoA 17Lu17015 rpoA 18

Example 2.5: Core Genome Comparisons and AAI Matrix

Genome comparisons have be performed using the software package EDGAR ofthe university GieBen (BMC Bioinformatics 10, 154, 2009;(https://edgar.computational.bio.uni-giessen.de/cgibin/edgar.cgi). Thedetermination of the core genome, the phylogenetic dendrograms on thebasis of the complete genome sequences and the AAI matrix values havebeen performed using the software package EDGAR. Results are shown inFIG. 22.

Example 3: Growth (Fermentability) of Strains for In-Vivo Tests

For green-house and field trials, the Paenibacillus strains were firstgrown on ISP2 plates (ready-to-use agar from BD [USA], catalog number277010). Afterwards, baffled shake flasks containing liquid ISP2 mediumwere inoculated with a colony from the agar plate and incubated for 5-7days at 150 rpm and 25° C. Depending on the test, either whole culturebroth, or the centrifuged and H₂O-washed cell pellet, or the supernatantwas applied to the plants. A scale-up to 10 L fermenters was possible.

Paenibacillus strains were grown in ISP2 liquid media (10 g/L maltextract, 4 g/L Bacto yeast extract, 4 g/L glucose monohydrate) for 6days at 22° C. at 150 rpm. OD_(600nm) indicating bacterial growth wasmeasured at different time points.

TABLE 5 Bacterial growth of Paenibacillus strains in liquid ISP2 medium.OD at 600 nm Paenibacillus strain 0 d 3 d 6 d Lu17007 0.011 3.110 3.050BD-62 0.013 0.442 0.446

Example 4—In-Vitro Confrontation Assay for Antifungal Activity

Antagonistic activity of the Paenibacillus strains against plantpathogens was shown in in-vitro confrontation assay. The phytopathogenicfungi used are Sclerotina sclerotiorum (SCLSCL), Botrytis cinerea(BOTRCI), Alternaria sp. (ALTESP) and Phytophthora infestans (PHYTIN).

As growth medium for BOTRCI, ALTESP, SCLSCL, ISP2 medium is usedcomprising per litre: 10 g malt extract (Sigma Aldrich, 70167); 4 gBacto yeast extract (Becton Dickinson, 212750); 4 g glucose monohydrate(Sigma Aldrich, 16301); 20 g Agar (Becton Dickinson, 214510), pH about7, Aq. bidest. As growth medium for PHYTIN, V8 medium is used comprisingper litre: 200 ml of vegetable juice, 3 g calcium carbonate (MerckMillipore, 1020660250); 30 g Agar (Becton Dickinson, 214510), pH 6.8,Aq. bidest.

The Paenibacillus strains were point-inoculated on one side of an agarplate. An agar block (approx. 0.3 cm²) containing one actively growingplant pathogen was put in the center of the plate. After incubating for7-14 days at 25° C., the growth of the plant pathogen was examined,especially for inhibition zones.

Thereafter, the agar plates are incubated at ° C. for about 7-14 daysbefore evaluation. Antibiosis is scored by evaluation of the diameter ofthe fungi-free zone (zone of inhibition). Competition is scored bycomparing the diameter of the growth of the fungal pathogen on plateswith bacterial strains in comparison to control plates. Mycoparasitismcan be documented in case the bacteria overgrows the fungal pathogen andalso parasitize the pathogens. This can be visualized by microscopy.

The novel Paenibacillus strains showed antifungal activity against alltested plant pathogens.

TABLE 6 In-vitro confrontation assay results. Diameter of zone ofinhibition [mm] Paenibacillus strain PHYTIN BOTRCI ALTESP SCLSCL Lu167748 2 2 2 Lu17007 8 8 5 2 Lu17015 8 5 5 2 BD-62 2 5 0 0

Example 5—Glasshouse Tests for Activity Against Plant Pathogenic Fungi

Use Example 5.1: Activity Against Late Blight on Tomato Caused byPhytophthora infestans with Protective Application

Commercially available young tomato seedlings (“Goldene Königin”) wereused for the described greenhouse trial. 2 replications (pots with 1plant each) were used per treatment. Plants were grown in commerciallyavailable substrate (Universal, Floragard) at approx. 22° C. in thegreenhouse. The humidity was controlled using a special device (˜90%humidity). The plants were sprayed to runoff with crude/whole culturebroth of 6 days old cultures of the respective Paenibacillus strain(depending on the setup) using a spray cabinet. Culture conditions forthe strains are described in Example 3. One day after application thetreated plants were inoculated with a suspension of sporangia ofPhytophthora infestans (PHYTIN). After inoculation, the trial plantswere immediately transferred to a humid chamber. The extent of fungalattack on the leaves was visually assessed 5-7 days after inoculation.Fungal attack in the untreated control was between 80-100% and set to100% for comparison reason.

TABLE 7 Paenibacillus strain PHYTIN (% fungal attack) Lu17007 4 Lu1677420 BD-62 53Use Example 5.2: Activity Against Grey Mold on Pepper Caused by Botrytiscinerea with Protective Application Commercially available young pepperseedlings (“Neusiedler Ideal”) were used for the described greenhousetrial. 2 replications (pots with 1 plant each) were used per treatment.Plants were grown in commercially available substrate (Universal,Floragard) at approx. 22° C. in the greenhouse. The humidity wascontrolled using a special device (˜90% humidity). The plants weresprayed to runoff with crude culture broth of 6 days old cultures of therespective Paenibacillus strain (depending on the setup) using a spraycabinet. Culture conditions for the strains are described in Example 3.One day after application the treated plants were inoculated with asuspension of spores of Botrytis cinerea (BOTRCI). After inoculation,the trial plants were immediately transferred to a humid chamber. Theextent of fungal attack on the leaves was visually assessed 5-7 daysafter inoculation. Fungal attack in the untreated control was between80-100% and set to 100% for comparison reason.

TABLE 8 Paenibacillus strain BOTRCI (% fungal attack) Lu17007 2 Lu1677416 Lu17015 20 BD-62 97Use Example 5.3: Activity Against Early Blight on Tomato Caused byAltemaria solani with Protective Application

Commercially available young tomato seedlings (“Goldene Königin”) wereused for the described greenhouse trial. 2 replications (pots with 1plant each) were used per treatment. Plants were grown in commerciallyavailable substrate (Universal, Floragard) at approx. 22° C. in thegreenhouse. The humidity was controlled using a special device (˜90%humidity). The plants were sprayed to runoff with crude/whole culturebroth of 6 days old cultures of the respective Paenibacillus strain(depending on the setup) using a spray cabinet. Culture conditions forthe strains are described in Example 3. One day after application thetreated plants were inoculated with a suspension of spores of Alternariasolani (ALTESO). After inoculation, the trial plants were immediatelytransferred to a humid chamber. The extent of fungal attack on theleaves was visually assessed 5-7 days after inoculation. Fungal attackin the untreated control was between 80-100% and set to 100% forcomparison reason.

TABLE 9 Paenibacillus strain ALTESO (% fungal attack) Lu17007 3 Lu1701516 BD-62 96Use Example 5.4: Activity Against Soybean Rust on Soybean Caused byPhakopsora pachyrhizi with Protective Application

Commercially available young soybean seedlings (“Mentor”) were used forthe described greenhouse trial. 2 replications (pots with 1 plant each)were used per treatment. Plants were grown in commercially availablesubstrate (Universal, Floragard) at approx. 22° C. in the greenhouse.The humidity was controlled using a special device (˜90% humidity). Theplants were sprayed to runoff with crude culture broth of 2-6 days oldcultures of Paenibacillus spp. (depending on the setup) using a spraycabinet. One day after application the treated plants were inoculatedwith a suspension of spores of Phakopsora pachyrhizi(PHAKPA). Afterinoculation, the trial plants were immediately transferred to a humidchamber. The extent of fungal attack on the leaves was visually assessed5-7 days after inoculation.

Use Example 5.5: Activity Against Fusarium Head Blight on Wheat Causedby Fusarium graminearum with Protective Application

Commercially available young wheat seedlings were used for the describedgreenhouse trial. 2 replications (pots with 1 plant each) were used pertreatment. Plants were grown in commercially available substrate(Universal, Floragard) at approx. 22° C. in the greenhouse. The humiditywas controlled using a special device (˜90% humidity). The plants weresprayed to runoff with crude culture broth of 2-6 days old cultures ofPaenibacillus spp. (depending on the setup) using a spray cabinet.Culture conditions are described in Example 3. One day after applicationthe treated plants were inoculated with a suspension of spores ofFusarium graminearum (GIBBZE). After inoculation, the trial plants wereimmediately transferred to a humid chamber. The extent of fungal attackon the leaves was visually assessed 5-7 days after inoculation.

Use Example 5.6: Activity Against Speckled Leaf Blotch on Wheat Causedby Septoria tritici with Protective Application

Commercially available young wheat seedlings were used for the describedgreenhouse trial. 2 replications (pots with 1 plant each) were used pertreatment. Plants were grown in commercially available substrate(Universal, Floragard) at approx. 22° C. in the greenhouse. The humiditywas controlled using a special device (˜90% humidity). The plants weresprayed to runoff with crude culture broth of 2-6 days old cultures ofPaenibacillus spp. (depending on the setup) using a spray cabinet.Culture conditions are described in Example 3. One day after applicationthe treated plants were inoculated with a suspension of spores ofSeptoria tritici (SEPTTR). After inoculation, the trial plants wereimmediately transferred to a humid chamber. The extent of fungal attackon the leaves was visually assessed 21-28 days after inoculation.

Use Example 5.7: Activity of the Paenibacillus Cells and of theSupernatant Against Various Pathogens with Protective Application

Whole culture broth from 6 days old cultures of Paenibacillus strainLu17007 was obtained according to Use Example 3 and used as in theexperimental setup of Use Example 5.1 to 5.3. Alternatively, such wholeculture broth was filtered through a filter with 0.2 μm pore size toobtain the culture medium and the crude cell fraction. The crude cellfraction could further be washed three times with the original volumesof phosphate-buffered saline to obtain washed cells.

The glasshouse trials were performed as described in the Use Examples5.1, 5.2 and 5.3 above for the respective pathogens Phytophthorainfestans, Botrytis cinerea and Alternaria solani. The extent of fungalattack on the leaves was visually assessed 5-7 days after inoculation.Fungal attack in the untreated control was between 80-100% and set to100% for comparison reason.

TABLE 10 Paenibacillus culture % fungal attack by component BOTRCIALTESO PHYTIN Whole culture broth 0 2 7 Culture medium 3 40 3 Crude cellfraction 0 5 4 Washed cells 1 10 1

Example 6—Enzymatic Tests

Use Example 6.1: Chitinase

Chitinase Test Solid Medium:

2 g/l NaNO₃, 1 g/l K₂HPO₄, 0.5 g/l MgSO₄, 0.5 g/l KCl, 0.2 g/l pepton,15 g/l agar, 10 g/l chitin from crab shells (Sigma-Aldrich C7170).

Test solid medium is autoclaved and filled into 9 cm Petri dishes.Paenibacillus strains are inoculated in the center of the plates andincubated for two days at 27° C. Thereafter, the plates are stained witha 1:3 diluted Lugol solution (Carl Roth N052.2) for 5 to 10 min. Lugolsolution is poured out and the plates are photographed and evaluated.Growth of the different strains was no more than 5-10 mm. Non-stainedzones (correlating with chitinase activity) varied from 0 mm (noactivity; “−” in Table 11) to several cm (“+” in Table 11).

Use Example 6.2: Cellulase

Cellulase Test Solid Medium:

2 g/l NaNO₃, 1 g/l K₂HPO₄, 0.5 g/l MgSO₄, 0.5 g/l KCl, 0.2 g/l pepton,15 g/l agar, carboxymethyl cellulose, sodium salt (Sigma-Aldrich419273).

Medium is autoclaved poured into 9 cm Petri dishes. Paenibacillusstrains are inoculated in the center of the plates and incubated for twodays at 27° C. After incubation plates are stained with a 1:3 dilutedLugol solution (Carl Roth N052.2) for 5 to 10 min. Lugol solution ispoured out and plates photographed.

Use Example 6.3: Amylase

Amylase Test Solid Medium:

2 g/l NaNO₃, 1 g/l K₂HPO₄, 0.5 g/l MgSO₄, 0.5 g/l KCl, 0.2 g/l pepton,15 g/l agar, 10 g/l soluble starch (Merck 1.01252).

Medium is autoclaved poured into 9 cm Petri dishes. Paenibacillusstrains are inoculated in the center of the plates and incubated for twodays at 27° C. After incubation plates are stained with a 1:3 dilutedLugol solution (Carl Roth N052.2) for 5 to 10 min. Lugol solution ispoured out and plates photographed.

TABLE 11 Chitinase, cellulose and amylase activities of Paenibacillusstrains. Strain Chitinase Cellulase Amylase Lu16774 + + − Lu17007 ++ + +Lu17015 + + + BD-62 − − − −, no activity; (+), low activity; +, regularactivity; ++, high activity.

Example 7—Fusaricidin-Type Metabolites Obtained from PaenibacillusStrains Example 7.1: Large Scale Cultivation of Bacterial Isolates andExtraction of Fusaricidin-Type Metabolites

a) Cultivation

The Paenibacillus strains were cultivated on agar plates containing GYMmedium (10 g/l glucose, 4 g/l yeast extract, 10 g/l malt extract; pH5.5, adjusted before autoclaving) and 20 g/l agar. Cultivation wasperformed for 10 to 20 days at room temperature. For maintenance agarslants with the same medium were used and stored at 4° C.

Small scale liquid cultures (250 ml GYM medium in 500 ml flasks) wereinoculated with 4-5 pieces of a well grown agar culture and cultivatedin an orbital shaker at 120 rpm at room temperature (20-23° C.).

Large scale fermentations were performed in 20 l fermenters with 15 lGYM medium (total capacity of fermenters was not used because of foamformation) inoculated with 250 ml well grown liquid culture andfermentation was carried out at room temperature (20-23° C.) withagitation (120 rpm) and aeration (3 l/min) for 5 to 8 days.

b) Extraction

One equal volume of isopropanol was added to the whole culture broth (noseparation of biomass from liquid culture was performed). Afteragitation and incubation for 2 to 16 hours, common table salt (sodiumchloride—100 to 200 g/l) was added to the mixture until phase separationof the organic and aqueous phase was visible.

The isopropanol phase was concentrated in vacuo. The resulting extract,still containing large amount of salt, was dissolved in methanol,centrifuged for better precipitation of salt residues, and the organicphase was concentrated again. This step was repeated until no saltprecipitate was present anymore.

c) Purification

i) Silica Gel Chromatography

30 grams of extract were dissolved in methanol and bound to 50 g silicagel (Merck, K60, 70-230 mesh), dried at 40° C. and layered onto 1 kg ofsilica gel (column 10 cm diameter, 30 cm high approx.).

Elution was carried out in four steps as following:

Step 1-4 l ethyl acetate

Step 2-4 l ethyl acetate:methanol (3:1, v/v)

Step 3-7 l ethyl acetate:methanol (1:1, v/v)

Step 4-4 l methanol

The third fraction (intermediate 1), containing the active compounds,was dried in vacuo and dissolved in 40% methanol (MeOH) in 0.1% formicacid (FA) (concentration: 100 mg/ml). The other fractions werediscarded.

ii) Chromabond HR-X Fractionation

20 ml of intermediate 1 was loaded onto a previously equilibrated (with40% MeOH in 0.1% FA) Chromabond HR-X cartridge (Macherey-Nagel, 1000 mg,ref 730941). The cartridge was washed with 100 ml 40% MeOH in 0.1% FAand eluted with 60 ml 70% MeOH in 0.1% FA. This intermediate 1-1 wasthen dried in vacuo.

iii) Preparative HPLC on a Sunfire C18 Column

Intermediate 1-1 was dissolved in DMSO (concentration: 200 mg/ml) and300 μl of intermediate 1-1 were chromatographed on a Sunfire C18 column(19×250 mm, 5 μm, Waters) as follows: 16 min at 10 ml/min, isocratic 70%0.2 FA; 30% acetonitrile (ACN), 1 min at 14 ml/min, gradient to 65% 0.2%FA; 35% ACN, 5 min at 14 ml/min, isocratic 65% 0.2% FA; 35% ACN.

Five fractions could be detected. All five resulting fractions weredried in vacuo and dissolved in DMSO (concentration: 125 mg/ml). Furtherpurification was performed using the same column and isocraticconditions (flow: 10.5 ml/min) adjusted for every fraction (12.5 mg perrun):

-   -   Fraction 1: 69% 0.2 FA; 31% ACN; two peaks detected (1-1 and        1-2)    -   Fraction 2: 69% 0.2 FA; 31% ACN; two peaks detected (2-1 and        2-2)    -   Fraction 3: 69% 0.2 FA; 31% ACN; three peaks detected (3-1, 3-2        and 3-3)    -   Fraction 4/5: 67% 0.2 FA; 33% ACN; one peak detected (4/5)    -   Fraction 6: 65% 0.2 FA; 35% ACN; two peaks detected (6-1 and        6-2)

The purity and quantity of the following samples was sufficient for NMRanalysis and structure elucidation: peaks 1-2, 2-1, 3-2, 4/5 and 6-1.

Example 7.2: Structural Elucidation of Novel Compounds 1A and 1B

From peak 2-1 of fraction 2, a mixture of compounds 1A and 1B (ratioabout 3:7) was obtained as a brown oil ([α]_(D) ²⁵=+20.9 (c=0.6,DMSO-d₆)).

The molecular formula C₄₇H₇₈N₁₀O₁₂ of the major component, compound 1B,was deduced from the HR-ESI-MS spectrum which gave a peak at m/z975.5863 [M+H]+; ESI-MS: 975.6 (100%, [M+H]*), 488.4 (51%, [M+2H]²⁺).

Besides, the mixture also contained as minor component, the lighterhomologue 1A, and the mass difference between both compounds was 14 amu.This observation was supported by a second peak observed in the ESI-MSspectrum at m/z 961.6.

The NMR spectra (Table 12) included in addition to signals ofexchangeable protons between δ 6.83 and 8.58, resonances of carbonyl inthe range of 5166.0-174.5 and methine signals between δ 47.8 and δ 60.4indicative for a peptide.

Extensive analysis of the 1D- and 2D-NMR data of compound 1B revealedthe presence of six amino acids including tyrosine (Tyr), glutamine(Gln), alanine (Ala), two threonines (Thr1 and Thr2) and isoleucine(Ile). Their sequence was found using two or three bonds correlationsacross amide functions. Thus, COSY, NOESY (FIG. 2) and HMBC (FIG. 3)spectra depicted correlations from the nitrogen-proton of Thr2 at δ 8.58to the signal of methine proton of Thr2 at δ 3.84 and the carbonyl at δ166.7 of Tyr while the same relationship was noted between thenitrogen-proton of Tyr at δ 8.52 and the signal of methylene proton ofTyr at δ 2.60 and the carbonyl at δ 170.4 of Ile. Furthermore, themethine hydrogen of Ile at δ 4.16 had a strong correlation with thecarbonyl signal of Ile at δ 170.4 and a weak contact with that of Thr1at δ 168.6; the signal of the β-methine proton at δ 5.30 of Thr1correlated with the carbonyl signal at δ 170.4 of Ala. Additionally tothe aforementioned correlations, others were displayed from the N-protonat δ 7.27 of Ala to the methine proton at δ 4.20 of the same amino acidwhile this latter proton had the same interaction with the carbonyl ofits amino and the one of Gln. Besides, a cross peak was revealed fromthe exchangeable proton at δ 8.20 of Gln to the methine hydrogen at δ3.87 of Gln and the carbonyl of Thr2 at δ 170.6; these above-mentioneddata suggested the cyclodepsipeptidic structure for compound 1B.

This cyclodepsipeptide 1B contained a terminal guanidine β-hydroxy fattyacid attached to Thr1 since a key correlation was observed between thesignal of its α-methine proton at δ 4.39 and the resonance of a carbonylat δ 171.9; HMBC contacts from that carbonyl at δ 171.9 to theamethylene protons at δ 2.35 and the β-methine proton at δ 3.77 werefurther observed as well as between the methylene protons at δ 3.03 andthe guanidine carbon at δ 157.2. The side chain was deduced to containtwelve methylene groups between the β-hydroxy and the guanidine group onthe basis of the fragment ion observed in the APCI-MS-MS spectrum of theparent [M+H]⁺ ion at m/z 256.2. Likewise, this spectrum providedinformation (FIG. 4b ) which confirmed the connection sequence of aminoacids and led to elucidate the structure of compound 1B as shown in FIG.1.

Signals of a CH₂ group at 2.80, 2.52/36.3 in the 1D- and 2D-spectracorresponded presumably to the β-CH₂ group of asparagine (Asn) incompound 1A. This conclusion was supported by reported data(Heterocycles 53, 1533-1549, 2000) in conjunction to fragments obtainedfrom MS/MS of the parent peak at m/z 961.6 (FIG. 4a ). Likewise, thelatter analyses provided information (FIGS. 4a, 4b ) which confirmed theconnection sequence of amino acids in both compounds and led toelucidate the structure of compounds 1A and 1B as shown in FIG. 1.

Example 7.3: Structural Identification of Compounds 2A and 2B asFusaricidins C and D

From peak 1-2 of fraction 1, a mixture of compounds 2A and 2B (ratioabout 1:1) was obtained as a brown oil. The molecular formula of theheavier component, compound 2B, was determined to be C₄₆H₇₆N₁₀O₁₂ on thebasis of the low resolution mass spectrometry. Analysis of the NMR data(Table 13) allowed to identify compound 2B as fusaricidin D. The lightercomponent of the mixture, compound 2A, was likewise identified asfusaricidin C, in which the Gin residue of fusaricidin C is replaced byAsn.

The mass spectrometric fragmentation pattern of the parent ions of m/z961.6 and 947.6 for compounds 2B and 2A, respectively, (FIGS. 5a, 5b )confirmed the length of the substituted fatty acid side chain to beidentical as in compound 1B. Fusaricidins C and D have formerly beenreported by Kajimura et al. (J. Antibiot. 50, 220-228, 1997).

Example 7.4: Structural Identification of Compound 3 as LI-F08b

From peak 6-1 of fraction 6, compound 3 was isolated as a brown oil andits low resolution presented a peak at m/z 925.6 [M+H]⁺ which, combinedwith NMR data (Table 14), led to the molecular formula C₄₄H₈₀N₁₀O₁₁.Compound 3 showed similar features in the NMR spectra as compound 1B andcompound 2B (fusaricidin D) except for the presence of aromatic signals(Table 14). Thus, characteristic resonances of a peptide were observednamely ten signals of protons attached to nitrogen between δ 6.89 and8.49, eight resonances of carbonyl ranged between δ 168.1 and 174.3, andsix signals of N-methine comprised between δ 48.0 and 59.5. A detailedanalysis of the HMQC, COSY and TOCSY spectra revealed the presence ofsix amino acids including Gln, two units of Thr, two units of Ile andAla. Furthermore, these spectra showed chemical shifts attributable tothe same β-hydroxyl fatty acid with a terminal guanidine as in compounds1A, 1B and fusaricidins C (2A) and D (2B). The position of this sidechain was determined on the basis of a long range correlation found onthe HMBC spectrum between the proton signal of N-methine at δ 4.44 ofThr1 and the carbonyl signal at δ 172.1 of the fatty acid. The sequenceof the amino acids was deduced from NOESY interactions and thefragmentation pattern (FIG. 6).

The combination of the NMR data (Table 14) and mass spectrometry led toidentify the metabolite compound 3 as LI-F08b, herein also calledfusaricidin LI-F08b, reported for the first time by Kuroda et al.(Heterocycles 53, 1533-1549, 2000).

Example 7.5: Structural Identification of Compounds 4A and 4B as LI-F06aand LI-F06b and of Compounds 5A and 5B as Fusaricidin A and B,Respectively

From peak 4/5 of fraction 4/5, a mixture of two further metabolites,compounds 4A and 4B (ratio about 1:3), was obtained which gave two peaksat m/z 897.5 (4A) and 911.6 (4B) in the ESI-MS spectrum, suggesting twofurther homologous cyclodepsipeptides. Resonances indicative forpeptides were observed in their NMR spectra (Table 15) as well as thoseof a β-hydroxyl fatty acid terminating in a guanidine group. Thefragmentation patterns of both parent ions found for compounds 4A and 4B(FIGS. 7a, 7b ) allowed to determine the sequence of amino acids and toidentify the constituents of the mixture as LI-F06a (4A) and LI-F06b(4B), respectively. Obtained from peak 3-2 of fraction 3, the mixture ofcompounds 5A and 5B (ratio about 1:3) was analyzed in the same manner.The ESI mass spectrum of the mixture showed two peaks at m/z 883.6 (5A)and 897.5 (5B) and the fragmentation patterns of these parent ions(FIGS. 8a, 8b ) in conjunction to NMR data (Table 16) allowed toidentify the components as fusaricidin A (5A) and fusaricidin B (5B).The data found for 4A, 4B, 5A and 5B matched those previously reported.(J. Antibiot. 50, 220-228, 1997; Heterocycles 53, 1533-1549, 2000).

TABLE 12 ¹H (DMSO-d₆, 600 MHz) and ¹³C-NMR (DMSO-d₆, 150 MHz) data ofcompounds 1A and 1B. Compounds 1 Compound 1A *Pos. δ_(H) δ_(C) Thr1 NH7.79 (br) — 1 — 168.6  2 4.46 (br d, 8.5) 56.4 3 5.30 (overlapped) 70.24 1.13 (overlapped) 16.6 Ala NH 7.22 (br) — 1 — nf* 2 4.13 (overlapped)47.7 3 1.11 (overlapped) 17.8 Asn NH 8.33 (overlapped) — 1 — 169.7  24.20 (1H, m) 50.6 3 2.52 (m), 2.80 (dd, 5.9, 15.1) 36.3 4 — 172.5  5 — —NH₂ 6.99 (br s), 7.42 (br s) — Thr2 NH 8.50 (overlapped) — 1 — 170.6  23.94 (m) 59.9 3 3.94 (m) 65.5 4 1.05 (br) 20.3 Tyr NH 8.48 (overlapped)— 1 — nf 2 4.60 (m) 54.2 3 2.60 (overlapped) 36.8 2.88 (overlapped) 4 —127.7  5 and 9 7.07 (d, 8.7) 130.2  6 and 8 6.60 (overlapped) 114.7  7 —155.9  Ile NH 7.28 (br s) — 1 — nf 2 4.16 (overlapped) 56.5 3 1.34(overlapped) 37.2 4 1.34 (overlapped) 25.4 5 0.52 (overlapped) 14.4 60.59 (overlapped) 11.4 *FA 1 — 171.9  2 2.35 (overlapped) 43.1 3 3.77(overlapped) 67.5 4 1.34 (overlapped) 36.8 5-12 1.19-1.30 (br s)29.0-29.2 13  1.25 (br s) 21.2 14  1.43 (overlapped) 28.7 15  3.03(overlapped) 40.6 *Gu NH nf — 16  — 157.2  Compound 1B Pos. δ_(H) δ_(C)Thr1 NH 8.18 (br s) — 1 — 168.6  2 4.39 (br d, 8.7) 56.9 3 5.30 (m) 70.24 1.13 (d, 6.4) 16.7 Ala NH 7.27 (br s) — 1 — 170.4  2 4.20 (m) 47.8 31.17 (d, 7.1) 17.8 Gln NH 8.20 (br s) — 1 — 170.4  2 3.87 (m) 53.2 31.96 (m), 2.08 (m) 26.2 4 2.08 (m), 2.18 (m) 32.0 — — 174.3  NH₂ 6.83(br s), 7.26(br s) — Thr2 NH 8.58 (br s) — 1 — 170.6  2 3.84 (m) 60.5 33.85 (m) 65.8 4 1.08 (overlapped) 20.0 Tyr NH 8.52 (br s) — 1 — 166.7  24.51 (m) 54.5 3 2.60 (m), 2.88 (m) 36.9 4 — 127.8  5 and 9 7.06 (d, 8.5)130.2  6 and 8 6.60 (d, 8.5) 114.7  7 — 155.9  Ile NH 7.42 (br s) — 1 —170.4  2 4.16 (br d, 8.5) 56.5 3 1.34 (m) 37.2 4 1.22 (m), 1.34 (m) 25.45 0.53 (overlapped) 14.4 6 0.61 (overlapped) 11.4 FA 1 — 171.9  2 2.35(m) 43.3 3 3.77 (m) 67.5 4 1.34 (m) 36.9 5-12 1.19-1.30 (br s) 29.0-29.213  1.25 (br s) 21.2 14  1.43 (m) 28.5 15  3.03 (q, 6.6) 40.6 Gu NH 8.40(br s) — 16  — 157.2 

TABLE 13 ¹H (DMSO-d₆, 600 MHz) and ¹³C-NMR (DMSO-d₆, 150 MHz) data ofcompounds 2A and 2B. Compounds 2 = fusaricidins C and D Compound 2A =fusaricidin C Pos. δ_(H) δ_(C) Thr1 NH 7.66 (d, 7.1) — 1 — 168.5  2 4.44(br d, 8.9) 56.6 3 5.31 (m) 70.2 4 1.13 (overlapped) 16.5 Ala NH 7.21(br) — 1 — nf 2 4.12 (m) 47.7 3 1.12 (overlapped) 17.8 Asn NH 8.26 (br)— 1 — 169.7  2 4.21 (m) 50.5 3 2.53 (overlapped), 2.80 (dd, 6.3, 15.0)36.3 4 — 172.6  5 — — NH₂ nf — Thr2 NH 8.52 (overlapped) — 1 — 170.3  23.85 (m) 60.5 3 3.86 (m) 65.8 4 1.09 (d, 5.7) 19.9 OH-3 4.96 (br d, 4.2)— Tyr NH 8.46 (overlapped) — 1 — nf 2 4.60 (m) 54.2 3 2.63 (overlapped)36.9 2.87 (overlapped) 4 — 127.7  5 and 9 7.08 (overlapped) 130.2  6 and8 6.60 (overlapped) 114.7  7 — 155.8  OH nf — Val NH 7.30 (overlapped) —1 — nf 2 4.12 (br s) 57.5 3 1.59 (m) 30.9 4 0.56 (d, 6.4) 18.2 5 0.35(d, 6.5) 18.7 FA 1 — nf 2 2.37 (overlapped) 43.1 3 3.79 (overlapped)67.5 4 1.35 (overlapped) 36.9 5 1.22 (overlapped) 25.3 6-12 1.20-1.27(br s) 29.1-29.2 13  1.26 (br s) 26.1 14  1.44 (overlapped) 28.5 15 3.07 (overlapped) 40.7 Gu NH nf — 16  — 156.8  Compound 2B = fusaricidinD Pos. δ_(H) δ_(C) Thr1 NH 8.17 (br s) — 1 — 168.6  2 4.40 (br d, 8.9)57.0 3 5.30 (m) 70.3 4 1.14 (overlapped) 16.7 Ala NH 7.60 (br s) 1 —170.6  2 4.19 (m) 47.8 3 1.17 (d, 7.2) 17.7 Gln NH 8.08 (br s) — 1 —170.4  2 3.86 (m) 53.2 3 1.98 (m), 2.09 (m) 26.1 4 2.10 (m), 2.18 (m)31.9 5 — 174.3  NH₂ 6.84 (br s), 7.28 (br s) — Thr2 NH 8.47 (overlapped)— 1 — 170.6  2 3.94 (m) 59.9 3 3.92 (m) 65.7 4 1.05 (d, 5.8) 20.2 OH-35.05 (d, 2.9) — Tyr NH 8.52 (overlapped) — 1 — 172.3  2 4.52 (m) 54.6 32.63 (m), 2.87(m) 36.9 4 — 127.7  5 and 9 7.06 (d, 8.4) 130.2  6 and 86.60 (d, 8.4) 114.7  7 — 155.8  OH 9.13 (br s) — Val NH 7.42 (br s) — 1— 170.3  2 4.12 (br s) 57.5 3 1.59 (m) 31.0 4 0.57 (d, 6.3) 18.3 5 0.40(d, 6.6) 18.7 FA 1 — 172.0  2 2.37 (m) 43.3 3 3.79 (m) 67.6 4 1.35 (m)36.9 5 1.22 (br s) 25.3 6-12 1.20-1.27 (br s) 29.1-29.2 13  1.26 (br s)26.1 14  1.44 (m) 28.7 15  3.07 (q, 6.7) 40.7 Gu NH 7.60 (br s) — 16  —156.8 

TABLE 14 ¹H (DMSO-d₆, 600 MHz) and ¹³C-NMR (DMSO-d₆, 150 MHz) data ofcompound 3 being LI-F08b. Compound 3 = LI-F08b Pos. δ_(H) δ_(C) Thr1 NH7.55 (br s) — 1 — 168.1  2 4.44 (br d, 8.4) 56.6 3 5.33 (m) 70.2 4 1.15(d, 6.5) 16.7 Ala NH 7.53 (br s) — 1 — 170.6  2 4.05 (m) 48.0 3 1.22 (brs) 17.2 Gln NH 7.93 (br s) — 1 — 170.5  2 3.94 (m) 52.7 3 1.98 (m), 2.09(m) 26.5 4 2.12 (m), 2.20 (m) 31.9 5 — 174.3  NH₂ 6.89 (br s), 7.32 (brs) — Thr2 NH 8.48 (br s) — 1 — 170.7  2 4.03 (m) 59.5 3 3.98 (m) 65.7 41.08 (d, 6.1) 19.8 Ile1 NH 8.49 (br s) — 1 — 172.5  2 4.15 (t, 7.6) 57.33 1.81 (m) 35.4 4 1.17 (m), 1.41 (m) 24.4 5 0.80 (t, 6.3) 10.6 6 0.81(d, 7.2) 15.5 Ile2 NH 7.30 (br s) — 1 — 171.3  2 4.53 (m) 55.3 3 1.65(m) 38.2 4 1.01 (m), 1.37 (m) 25.5 5 0.83 (t, 6.4) 11.4 6 0.70 (d, 7.4)14.2 FA 1 — 172.1  2 2.37 (d, 5.7) 43.4 3 3.77 (m) 67.6 4 1.37 (m) 36.95-12 1.20-1.28 (br s) 29.0-29.2 13  1.25 (br s) 26.2 14  1.43 (m) 28.715  3.03 (q, 6.7) 40.6 Gu NH 8.37 (br s) — 16  — 157.2 

TABLE 15 ¹H (DMSO-d₆, 600 MHz) and ¹³C-NMR (DMSO-d₆, 150 MHz) data ofcompounds 4A and 4B. Compounds 4 = LI-F06a and LI-F06b Compound 4A =LI-F06a Pos. δ_(H) δ_(C) Thr1 NH 8.31 (br) — 1 — 168.5  2 4.40 (m) 56.93 5.30 (m) 70.5 4 1.14 (m) 16.6 Ala NH nf — 1 — 170.6  2 3.97 (m) 47.9 31.15 (overlapped) 17.3 Asn NH 8.06 (br) — 1 — 169.8  2 4.28 (m) 50.5 32.55 (m), 2.75 (dd, 6.7, 15.1) 36.9 4 — 172.6  5 — — NH₂ nf — Thr2 NH8.54 (br) 1 — 170.4  2 3.91 (m) 60.5 3 3.92 (m) 65.6 4 1.09 (d, 6.4)19.6 Val NH 7.28 (m) — 1 — nf 2 4.40 (overlapped) 57.3 3 1.83(overlapped) 32.0 4 0.75 (d, 6.6) 18.1 5 0.84 (overlapped) 19.3 Ile NH7.31 (overlapped) — 1 — nf 2 4.51 (overlapped) 55.5 3 1.65 (overlapped)38.1 4 1.02 (m), 1.36 (m) 25.4 5 0.82 (overlapped) 15.6 6 0.72(overlapped) 14.4 FA 1 — 172.1  2 2.44 (dd) 43.1 3 3.81 (m) 67.7 4 1.37(overlapped) 36.9 5-12 1.22-1.24 (br s) 29.1-29.2 13  1.25 (br s) 26.414  1.43 (m) 28.5 15  3.03 (q, 6.7) 40.7 Gu NH nf — 16  — 157.2 Compound 4B = LI-F06b Pos. δ_(H) δ_(C) Thr1 NH 7.59 (br s) — 1 — 168.4 2 4.44 (m) 56.7 3 5.32 (m) 70.3 4 1.15 (m) 16.6 Ala NH 7.53 (br s) 1 —170.7  2 4.07 (m) 48.0 3 1.21 (d, 7.3) 17.4 Gln NH 7.96 (br s) — 1 —170.7  2 3.93 (m) 52.9 3 1.97 (m), 2.10 (m) 26.5 4 2.12 (m), 2.21 (m)32.0 5 — 174.4  NH₂ 6.88 (br s), 7.33 (br s) — Thr2 NH 8.48 (br) — 1 —170.6  2 4.02 (m) 59.7 3 3.99 (m) 65.7 4 1.08 (d, 6.4) 19.8 Val NH 7.39(m) — 1 — 171.0  2 4.39 (m) 57.0 3 1.83 (m) 31.6 4 0.74 (d, 6.6) 18.4 50.80 (overlapped) 19.2 Ile NH 7.23 (overlapped) — 1 — 171.2  2 4.51 (1H,m) 55.6 3 1.65 (m) 38.1 4 1.02 (m), 1.36 (m) 25.5 5 0.82 (overlapped)15.6 6 0.71 (overlapped) 14.3 FA 1 — 172.2  2 2.37 (m) 43.4 3 3.78 (m)67.7 4 1.37 (m) 36.9 5 1.22-1.24 (br s) 29.1-29.2 13  1.25 (br s) 26.214  1.43 (m) 28.5 15  3.03 (q, 6.7) 40.7 Gu NH 8.34 (br s) — 16  —157.2 

TABLE 16 ¹H (DMSO-d₆, 600 MHz) and ¹³C-NMR (DMSO-d₆, 150 MHz) data ofcompounds 5A and 5B. Compounds 5 = fusaricidins A and B, LI-F04a andLI-F04b Compound 5A = fusaricidin A Pos. δ_(H) δ_(C) Thr1 NH 7.66 (br) —1 — 168.5  2 4.46 (m) 56.6 3 5.32 (m) 70.4 4 1.16 (overlapped) 16.3 AlaNH 7.26 (br) — 1 — 170.6  2 4.00 (m) 47.8 3 1.15 (overlapped) 17.4 AsnNH 8.10 (br) — 1 — 169.8  2 4.28 (q, 6.6) 50.5 3 2.53 (m), 2.76 (dd,6.6, 15.0) 36.7 4 — 172.5  5 — — NH₂ nf — Thr2 NH 8.54 (br s) — 1 — nf 23.91 (m) 60.4 3 3.91 (m) 65.6 4 1.09 (d, 5.6) 19.6 Val NH nf — 1 — nf 24.40 (m) 57.1 3 1.82 (m) 31.4 4 nf nf 5 0.82 (d, 6.0) 19.1 Val NH 8.41(br s) — 1 — 172.1  2 4.13 (m) 58.3 3 2.02 (m) 29.7 4 0.86 (d, 6.7) 18.25 0.84 (7.0) 19.3 FA 1 — 172.1  2 2.37 (br d, 5.8) 43.4 3 3.80 (m) 67.64 1.37 (m) 36.9 5-12 1.22-1.25 (br s) 26.2-29.2 13  1.25 (br s) 26.2 14 1.43 (m) 28.7 15  3.03 (q, 6.7) 40.6 Gu NH nf — 16  — 157.2  Compound 5B= fusaricidin B Pos. δ_(H) δ_(C) Thr1 NH 8.30 (d, 8.0) — 1 — 168.4  24.40 (br d, 8.5) 57.0 3 5.31 (m) 70.3 4 1.15 (d, 5.7) 16.6 Ala NH 7.53(br s) — 1 — 170.6  2 4.10 (m) 47.9 3 1.20 (d, 7.2) 17.5 Gln NH 8.53 (d,4.3) — 1 — 170.6  2 3.92 (m) 52.9 3 1.98 (m), 2.09 (m) 26.4 4 2.10 (m),2.20 (m) 31.9 5 — 174.3  NH₂ 6.86 (br s), 7.30 (br s) — Thr2 NH 8.46 (d,6.9) — 1 — 170.5  2 4.02 (m) 59.7 3 3.99 (m) 65.5 4 1.07 (d, 6.0) 19.9Val NH 7.29 (br s) — 1 — 171.2  2 4.40 (m) 57.1 3 1.82 (m) 31.5 4 0.76(d, 6.6) 18.4 5 0.81 (d, 6.2) 19.1 Val NH 8.37 (d, 7.6) — 1 — 173.1 24.23 (m) 57.8 3 1.99 (m) 30.2 4 0.86 (d, 6.7) 18.2 5 0.84 (7.0) 19.3 FA1 — 172.0  2 2.34 (dd, 7.0, 13.5), 2.44 (dd, 4.9, 13.5) 43.4 3 3.80 (m)67.6 4 1.37 (m) 36.8 5-12 1.22-1.25 (br s) 26.2-29.2 13  1.25 (br s)26.2 14  1.43 (m) 28.5 15  3.03 (q, 6.7) 40.6 Gu NH 8.47 (br s) — 16  —157.2 

No hydrolysis experiments were carried out to determine theconfiguration of the constituting amino acids.

Example 8—Metabolites Produced by Paenibacillus Strains Example 8.1:Production of Metabolites by Paenibacillus Strains

The presence of fusaricidins in general and in particular of the knownfusaricidins A, B, C, D, LI-F06a, LI-F06b and LI-F08b as well as thenovel fusaricidin-type compounds 1A and 1B was determined for thePaenibacillus strains following the procedural steps which are describedin Example 7.1 above.

TABLE 17 Fusaricidin-type metabolite production of the Paenibacillusstrains. Compound/Fusaricidin 2A 2B 3 4A 4B 5A 5B Strains 1A 1B C DLI-F08b LI-F06a LI-F06b A B Lu16774 + ++ ++ ++ ++ − − ++ ++ Lu17007 + ++++ ++ ++ + ++ ++ ++ Lu17015 ++ ++ ++ ++ ++ ++ ++ ++ ++ BD-62 − − − − − −− − − Legend: −, compound not detectable; +, compound detectable; ++compound detectable at higher amounts compared to scale +.

The whole culture broth of all of the novel Paenibacillus strainsLu16774, Lu17007 and Lu17015 contained at least one the fusaricidin-typemetabolites identified in Example 7 (Table 17). None of thesefusaricidin-type metabolites were detected in the whole culture broth ofP. peoriae strain BD-62.

The whole culture broth of the novel Paenibacillus strains Lu16774,Lu17007 and Lu17015 all contained the novel fusaricidin-type compounds1A and 1B. Further, the whole culture broth of the novel Paenibacillusstrains Lu16774, Lu17007 and Lu17015 all contained the fusaricidins A,B, C and D as well as LI-F08b. In addition, the whole culture broth ofthe novel Paenibacillus strains Lu17007 and Lu17015 containedfusaricidins LI-F06a and LI-F06b.

Compounds 1A and 1B were not detected in the whole culture broth of theclosely related P. peoriae strain BD-62. Fusaricidins A, B, C and D,LI-F06a, LI-F06b and LI-F08b were also not in the whole culture broth ofP. peoriae strain BD-62.

Example 9: Activity of Metabolites by Paenibacillus Strains AgainstVarious Fungal Pathogens

The compounds 1A and 1B, fusaricidin A, B and D and were obtained wereused in the following experiments.

Fungal growth assays were performed in 96 well plates with sporesuspension of the pathogen Botrytis cinerea (BOTRCI, in YBA [10 g Bactopeptone (Becton Dickinson 211677), 10 g yeast extract (Becton Dickinson212750), 20 g sodium acetate, ad 1000 mL aqua bidest] or Alternariasolani (ALTESO, in YBG [10 g Bacto peptone (Becton Dickinson 211677), 10g yeast extract (Becton Dickinson 212750), 20 g glycerine 99%, ad 1000mL aqua bidest]). Fusaricidins and compounds 1A and 1B were dissolvedand diluted in DMSO. Different concentrations ranging from 60 μM down to0.3 μM were pipetted into the microtiter plate. An aqueous suspension of10⁴ spores/ml was added. The plates were incubated at about 18° C.Fungal growth was determined by measuring the optical density at 600 nmin a microplate reader 3 and 7 days after the inoculation of the sporesand compared to the untreated control (DMSO). IC₅₀ (concentration [μM]of the respective metabolite required for 50% inhibition of fungalgrowth) has been determined thereafter.

Notably, the compounds 1A and 1B showed the highest antifungal efficacywith IC₅₀ values of 0.4-0.6 μM (Tab. 18).

TABLE 18 Antifungal growth inhibition of Paenibacillus metabolites IC₅₀values Compound/Fusaricidin 2B 5A 5B Pathogen 1A 1B Fus. D Fus. A Fus. B(Evaluation day) Fungal growth inhibition (IC50 [μM]) ALTESO (3 d) 0.60.6 1.1 1.3 1.1 ALTESO (7 d) 0.5 0.4 0.6 0.7 0.6 BOTRCI (7 d) 0.3 0.40.5 0.5 0.6 “—” means that growth inhibition in tested concentrationrange not sufficient to determine IC₅₀.

In addition, glasshouse trials were performed with Compounds 1A and 1Bas described in the Use Examples 5.1 to 5.5 above for the respectivepathogens Botrytis cinerea (BOTRCI), Alternaria solani(ALTESO),Phytophthora infestans (PHYTIN), Phakopsora pachyrhizi (PHAKPA) andFusarium graminearum (GIBBZE). The extent of fungal attack on the leaveswas visually assessed 5-7 days after inoculation.

Notably, compounds 1A and 1B were effective in controlling importantfungal diseases on crop plants already at dose levels as low as 7.2 ppmand showed higher antifungal efficacy than Fusaricidin A, B and D(Tables 19 to 21).

TABLE 19 Antifungal efficacy of metabolites determined in planta. %efficacy (% fungal attack) Metabolite tested Conc. BOTRCI ALTESO PHYTINPHAKPA GIBBZE Untreated — 0 (100) 0 (100) 0 (100) 0 (100) 0 (100)Compound 1A 360 ppm 99 100 95 49 Compound 1A  36 ppm 97 74 Compound 1B360 ppm 100 100 97 Compound 1B  36 ppm 97

TABLE 20 Efficacy of metabolites against late blight on tomato caused byPhytophthora infestans with protective application. % efficacyMetabolite tested Conc. (% fungal attack) Untreated — 0 (100)Fusaricidin A 7.2 ppm 15 Fusaricidin B 7.2 ppm 4 Fusaricidin D 7.2 ppm 0Compound 1B 7.2 ppm 44

TABLE 21 Efficacy of metabolites against head blight on wheat caused byFusarium graminearum with protective application. % efficacy Metabolitetested Conc. (% fungal attack) Untreated — 0 (100) Fusaricidin A 360 ppm31 Fusaricidin B 360 ppm 0 Compound 1A 360 ppm 49

TABLE 22 Efficacy of metabolites against head blight on wheat caused bySeptoria tritici with protective application. % efficacy Metabolitetested Conc. (% fungal attack) Untreated — 0 (100) Fusaricidin D 360 ppm50 Compound 1B 360 ppm 80

Example 10: Comparison of Activity of Paenibacillus polymyxa nov. ssp.Plantarum Strains Lu16674 and Lu17007 of According to the Invention withPaenibacillus polymyxa nov. ssp. Plantarum M-1 Against Various Pathogensin Glass House Trials

Whole culture broth from 6 days old cultures of Paenibacillus strainLu17007, Lu16674 and M1 was obtained according to Use Example 3 and usedas in the experimental setup of Use Example 5.1 to 5.5. The glasshousetrials were performed as described in the Use Examples 5.1 to 5.5 abovefor the respective pathogens. The extent of fungal attack on the leaveswas visually assessed 5-7 days after inoculation.

Notably, the Paenibacillus strains Lu16774 and Lu17007 were effective incontrolling important fungal diseases on crop plants even at highdilution factors and showed higher antifungal efficacy than the closelyrelated strain M-1 (Tables 22 to 27).

TABLE 22 Paenibacillus Dilution factor of BOTRCI % efficacy strain wholeculture broth (% fungal attack) Untreated 0 (100) Lu16674 1:10 95 M-11:10 86 Lu16674 1:50 76 Lu17007 1:50 98 M-1 1:50 51

TABLE 23 Paenibacillus Dilution factor of BOTRCI % efficacy strain wholeculture broth (% fungal attack) Untreated 0 (100) Lu17007 undiluted 92M-1 undiluted 87 Lu17007 1:10 84 M-1 1:10 53 Lu17007 1:50 63 M-1 1:50 32

TABLE 24 Paenibacillus Dilution factor of ALTESO % efficacy strain wholeculture broth (% fungal attack) Untreated 0 (100) Lu16674 1:10 77 M-11:10 41

TABLE 25 Paenibacillus Dilution factor of PHYTIN % efficacy strain wholeculture broth (% fungal attack) Untreated 0 (100) Lu17007 1:10 83 M-11:10 42 Lu16674 1:50 13 Lu17007 1:50 30 M-1 1:50 0

TABLE 26 Paenibacillus Dilution factor of PHAKPA % efficacy strain wholeculture broth (% fungal attack) Untreated 0 (100) Lu17007 undiluted 94M-1 Undiluted 87

TABLE 27 Paenibacillus Dilution factor of GIBBZE % efficacy strain wholeculture broth (% fungal attack) Untreated 0 (100) Lu17007 undiluted 70M-1 undiluted 31 Lu16674 1:50 52 Lu17007 1:50 33 M-1 1:50 24

The documents as cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Compounds 1A, 1B, 2A, 2B, 3, 4A, 4B, 5A and 5B.

FIG. 2. Key NOESY and COSY correlations of compound 1B.

FIG. 3. HMBC correlation of compound 1B.

FIG. 4. Fragmentation patterns a) of compound 1A and b) of compound 1B.

FIG. 5. Fragmentation patterns a) of compound 2A (fusaricidin C) and b)of compound 2B (fusaricidin D).

FIG. 6. Fragmentation pattern of compound 3 (LI-F08b).

FIG. 7. Fragmentation patterns a) of compound 4A (LI-F06a) and b) ofcompound 4B (LI-F06b).

FIG. 8. Fragmentation patterns a) of compound 5A (fusaricidin A) and b)of compound 5B (fusaricidin B).

FIG. 9 shows the percentage identity of the complete 16S rDNA sequenceof the Paenibacillus strains of the invention to related taxa aftermultiple sequence alignment.

Legend: * Strain numbers: 1=Paenibacillus strain Lu16774;2=Paenibacillus strain Lu17015; 3=Paenibacillus strain Lu17007;4=Paenibacillus peoriae NRRL BD-62; 5=Paenibacillus anaericanus MH21;6=Paenibacillus brasiliensis PB172; 7=Paenibacillus campinasensis 324;8=Paenibacillus chibensis JCM 9905; 9=Paenibacillus glucanolyticus DSM5162; 10=Paenibacillus hunanensis FeL05; 11=Paenibacillus jamilae CECT5266; 12=Paenibacillus kribbensis AM49; 13=Paenibacillus lactis MB 1871;14=Paenibacillus lautus JCM 9073; 15=Paenibacillus macerans IAM 12467;16=Paenibacillus massiliensis 2301065; 17=Paenibacillus pabuli HSCC 492;18=Paenibacillus peoriae DSM 8320 (BD-57); 19=Paenibacillus pini S22;20=Paenibacillus polymyxa IAM 13419; 21=Paenibacillus purispatiiES_MS17; 22=Paenibacillus sediminis GT-H3; 23=Paenibacillus terraeAM141; 24=Paenibacillus terrigena A35; 25=Paenibacillus timonensis2301032; 26=Paenibacillus turicensis MOL722; 27=Paenibacillus uliginisN3/975; 28=Cohnella thermotolerans CCUG 47242. Strains 6 to 28 are typestrains for the respective species.

Similarities of the novel strains with Paenibacillus peoriae (NRRL BD-62and DSM 8320) have been marked in bold letters.

FIG. 10 shows a phylogenetic dendrogram calculated from the % identityof 16S-rDNA sequences of the Paenibacillus strains of the invention withother taxa (FIG. 9). The root of the tree was determined by includingthe 16S rRNA gene sequence of Cohnella thermotolerans into the analysis.The scale bar below the dendrogram indicates 1 nucleotide substitutionsper 100 nucleotides.

FIG. 11 shows the RiboPrint pattern obtained from samples of thePaenibacillus strains of the invention in comparison to a sample of theclosely related P. peoriae strain BD-62 using RiboPrinter MicrobialCharacterization System and a phylogenetic dendrogram resultingtherefrom.

FIG. 12 shows the percentage identity of the DNA sequence of thednaNgene of the Paenibacillus strains of the invention to relatedPaenibacillus strains after multiple sequence alignment. Legend: *Strain numbers: 1=Paenibacillus strain Lu16774; 2=Paenibacillus strainLu17007; 3=Paenibacillus strain Lu17015; 4=P. peoriae DSM 8320^(T)=KCTC3763^(T) (GenBank acc. no. AGFX00000000; J. Bacteriol. 194, 1237-1238,2012); 5=P. polymyxa 1-43 (GenBank acc. no. ASRZ01000000; deposition no.GCMCC 4965; CN 102352332 B); 6=P. polymyxa A18 (GenBank acc. noJWJJ00000000.1; NCBI Project ID 225496); 7=P. polymyxa ATCC 842^(T)=DSM36^(T)=KCTC 3858^(T) (GenBank acc. no. AFOX00000000; J. Bacteriol.193(18), 5026-5027, 2011); 8=P. polymyxa CF05 (GenBank acc. no.CP009909; Genome Announc 3(2):e00198-15. Doi:10.1128/genomeA.00198-15);9=P. polymyxa CICC 10580 (GenBank acc. no. JNCB00000000; Genome Announc.2(4):e00854-14. doi:10.1128/genomeA.00854-14); 10=P. polymyxa DSM 365(GenBank acc. no. JMIQ00000000; J. Biotechnol. 195, 72-73, 2015); 11=P.polymyxa E681 (GenBank acc. no. CP000154; GenomeNet Ref Seq NC_014483.2;J. Bacteriol. 192(22), 6103-6104, 2010); 12=P. polymyxa M-1 (GenBankacc. no. HE577054.1; GenomeNet Ref Seq NC_017542.1); 13=P. polymyxa NRRLB-30509 (GenBank acc. no. JTH000000000; Genome Announc. 2015March-April; 3(2): e00372-15); 14=P. polymyxa SC2 (GenBank acc. no.CP002213; J. Bacteriol. 193 (1), 311-312, 2011); 15=P. polymyxa SQR-21(GenBank acc. no. CP006872; GenomeNet Ref Seq NZ_CP006872.1; GenomeAnnounc. 2014 March-April; 2(2): e00281-14); 16=P. polymyxa Sb3-1(GenBank acc. no. CP010268; Genome Announc. 2015 March-April; 3(2):e00052-15); 17=P. polymyxa TD94 (GenBank acc. no. ASSA00000000); 17=P.polymyxa WLY78 (GenBank acc. no. ALJV00000000); P. terrae HPL-003(GenBank acc. no. CP003107; NCBI Ref Seq NC_016641.1); P. polymyxa CR1(GenBank acc. no. CP006941; Genome Announc. 2014 January-February; 2(1):e01218-13).

FIG. 13 shows the percentage identity of the DNA sequence of thecomplete gyrB gene of the Paenibacillus strains of the invention torelated Paenibacillus strains after multiple sequence alignment. Strainnumbers are described in Legend to FIG. 12.

FIG. 14 shows the percentage identity of the DNA sequence of thecomplete recF gene of the Paenibacillus strains of the invention torelated Paenibacillus strains after multiple sequence alignment. Strainnumbers are described in Legend to FIG. 12.

FIG. 15 shows the percentage identity of the DNA sequence of thecomplete recN gene of the Paenibacillus strains of the invention torelated Paenibacillus strains after multiple sequence alignment. Strainnumbers are described in Legend to FIG. 12.

FIG. 16 shows the percentage identity of the DNA sequence of thecomplete rpoA gene of the Paenibacillus strains of the invention torelated Paenibacillus strains after multiple sequence alignment. Strainnumbers are described in Legend to FIG. 12.

FIG. 17 shows the maximum likelihood denrogram on basis of the completednaN gene sequence of strains of the P. polymyxa complex. The scale of0.1 shown corresponds to 1% nucleotide exchanges.

FIG. 18 shows the maximum likelihood denrogram on basis of the completegyrB gene sequence of strains of the P. polymyxa complex. The scale of0.1 shown corresponds to 1% nucleotide exchanges.

FIG. 19 shows the maximum likelihood denrogram on basis of the completerecF gene sequence of strains of the P. polymyxa complex. The scale of0.1 shown corresponds to 1% nucleotide exchanges.

FIG. 20 shows the maximum likelihood denrogram on basis of the completerecN gene sequence of strains of the P. polymyxa complex. The scale of0.1 shown corresponds to 1% nucleotide exchanges.

FIG. 21 shows the maximum likelihood denrogram on basis of the completerpoA gene sequence of strains of the P. polymyxa complex. The scale of0.1 shown corresponds to 1% nucleotide exchanges.

FIG. 22 shows the Amino Acid Index (AAI) matrix of representativegenomes of the P. polymyxa complex performed according to Example 2.5.Strain numbers are described in Legend to FIG. 12.

The invention claimed is:
 1. A method of controlling, suppressing plantpathogens or preventing plant pathogen infection, wherein the plantpathogens, their habitat or plants to be protected against plantpathogen attack, or the soil or propagation material are treated with aneffective amount of a composition comprising a) a Paenibacillus strain,which is selected from the group consisting of: i) strain Lu16774deposited with DSMZ under Accession No. DSM 26969; ii) strain Lu17007deposited with DSMZ under Accession No. DSM 26970; iii) strain Lu17015deposited with DSMZ under Accession No. DSM 26971; and iv) a strainwhich comprises a DNA sequence exhibiting (1) at least 99.6% nucleotidesequence identity to the DNA sequences SEQ ID NO:4 or SEQ ID NO:9; or(2) at least 99.8% nucleotide sequence identity to the DNA sequence SEQID NO:14; or (3) at least 99.9% nucleotide sequence identity to the DNAsequences SEQ ID NO:5 or SEQ ID NO:10; or (4) at least 99.2% nucleotidesequence identity to the DNA sequence SEQ ID NO:15; or (5) at least99.2% nucleotide sequence identity to the DNA sequences SEQ ID NO:6 orSEQ ID NO:11; or (6) at least 99.8% nucleotide sequence identity to theDNA sequence SEQ ID NO:16; or (7) at least 99.8% nucleotide sequenceidentity to the DNA sequences SEQ ID NO:7 or SEQ ID NO:12; or (8) atleast 99.3% nucleotide sequence identity to the DNA sequence SEQ IDNO:17; or (9) 100.0% nucleotide sequence identity to the DNA sequencesSEQ ID NO:8 or SEQ ID NO:13; or (10) 100% nucleotide sequence identityto the DNA sequence SEQ ID NO:18; or b) a substantially purified cultureof the Paenibacillus strain; or c) a whole culture broth or a cell-freeextract of the Paenibacillus strain; or d) a compound of formula I

wherein R is selected from 15-guanidino-3-hydroxypentadecanoic acid and12-guanidinododecanoic acid; X¹ is threonine; X² is isoleucine; X³ istyrosine; X⁴ is threonine; X⁵ is selected from glutamine and asparagine;X⁶ is alanine; and wherein an arrow defines a single amide bond eitherbetween the carbonyl moiety of R and the amino group of the amino acidX¹ or between the carbonyl group of one amino acid and the amino groupof a neighboring amino acid, wherein the tip of the arrow indicates theattachment to the amino group of said amino acid X¹ or of saidneighboring amino acid; and wherein the single line without an arrowhead defines a single ester bond between the carbonyl group of X⁶ andthe hydroxyl group of X¹; or an agriculturally acceptable salt thereof;and an auxiliary.
 2. The method of claim 1, wherein the plant pathogensand/or harmful microorganisms are selected from harmful fungi.